Bioproduction of astaxanthin using mutant carotenoid ketolase and carotenoid hydroxylase genes

ABSTRACT

Protein engineered nucleic acid fragments encoding a CrtO ketolase and a CrtZ hydroxylase are provided with increased astaxanthin synthesis activity. Methods using the present nucleic acid fragments are also provided for increasing or altering astaxanthin production in suitable production hosts.

FIELD OF THE INVENTION

This invention is in the field of microbiology, molecular biology, andthe use of carotenoid ketolases and carotenoid hydroxlyases to produceastaxanthin. More specifically, nucleic acid molecules encoding both aCrtO carotenoid ketolase and a CrtZ carotenoid hydroxylase are providedthat are characterized by improved astaxanthin production. Methods forrecombinant production of astaxanthin using the present nucleic acidmolecules are also provided.

BACKGROUND OF THE INVENTION

Carotenoids are pigments that are ubiquitous throughout nature andsynthesized by all photosynthetic organisms, and in some heterotrophicgrowing bacteria and fungi. Carotenoids provide color for flowers,vegetables, insects, fish and birds. Colors of carotenoid range fromyellow to red with variations of brown and purple. As precursors ofvitamin A, carotenoids are fundamental components in our diet and theyplay additional important role in human health. Industrial uses ofcarotenoids include pharmaceuticals, food supplements, animal feedadditives and colorants in cosmetics to mention a few.

Because animals are unable to synthesize carotenoids de novo, they mustobtain them by dietary means. Thus, manipulation of carotenoidproduction and composition in plants or bacteria can provide new orimproved source for carotenoids.

Carotenoids come in many different forms and chemical structures. Mostnaturally occurring carotenoids are hydrophobic tetraterpenoidscontaining a C₄₀ methyl-branched hydrocarbon backbone derived fromsuccessive condensation of eight C₅ isoprene units (IPP). In addition,rare carotenoids with longer or shorter backbones occur in some speciesof nonphotosynthetic bacteria. The term “carotenoid” actually includeboth carotenes and xanthophylls. A “carotene” refers to a hydrocarboncarotenoid. Carotene derivatives that contain one or more oxygen atoms,in the form of hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydicfunctional groups, or within glycosides, glycoside esters, or sulfates,are collectively known as “xanthophylls”. Carotenoids are furthermoredescribed as being acyclic, monocyclic, or bicyclic depending on whetherthe ends of the hydrocarbon backbones have been cyclized to yieldaliphatic or cyclic ring structures (G. Armstrong, (1999) InComprehensive Natural Products Chemistry, Elsevier Press, volume 2, pp321-352).

Carotenoid biosynthesis starts with the isoprenoid pathway and thegeneration of a C5 isoprene unit, isopentenyl pyrophosphate (IPP). IPPis condensed with its isomer dimethylallyl pyrophophate (DMAPP) to formthe C10, geranyl pyrophosphate (GPP), and elongated to the C15, farnesylpyrophosphate (FPP). FPP synthesis is common to both carotenogenic andnon-carotenogenic bacteria. Enzymes in subsequent carotenoid pathwaysgenerate carotenoid pigments from the FPP precursor and can be dividedinto two categories: carotene backbone synthesis enzymes and subsequentmodification enzymes. The backbone synthesis enzymes include geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoenedehydrogenase and lycopene cyclase, etc. The modification enzymesinclude ketolases, hydroxylases, dehydratases, glycosylases, etc. Unlikegenes in the upstream isoprenoid pathway that are common in allorganisms, the downstream carotenoid modifying enzymes are less common.

Carotenoid hydroxylases are a class of enzymes that introduce hydroxylgroups to the ionone ring of the cyclic carotenoids, such as β-carotene,echinenone, 3′-hydroxyechinenone, β-cryptoxanthin, adonirubin, andcanthaxanthin to produce hydroxylated carotenoids. Examples of suchcarotenoids include astaxanthin, β-cryptoxanthin, zeaxanthin,3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin, adonixanthin,tetrhydroxy-β,β′-caroten-4,4′-dione, tetrahydroxy-β,β′-caroten-4-one,caloxanthin, erythroxanthin, nostoxanthin, flexixanthin,3-hydroxy-γ-carotene, 3-hydroxy-4-keto-γ-carotene, bacteriorubixanthin,bacteriorubixanthinal, and lutein.

Several classes of carotenoid hydroxylases have been reported (i.e.CrtR-type and CrtZ-type). Both CrtR and CrtZ enzymes catalyze additionof hydroxyl groups to the β-ionone rings of cyclic carotenoids. However,no significant sequence homology exists between CrtR hydroxylases andthe CrtZ hydroxylases. The CrtR-type carotenoid hydroxylases have beenreported in Cyanobacteria such as Synechocystis sp. PCC 6803 (Lagarde,D., and Vermaas, W., FEBS Lett., 454(3):247-251 (1999) and in plants.The CrtZ-type carotenoid hydroxylases have been reported from a varietyof bacterial, fungal, algal, and plant species. Examples include, butare not limited to, bacterial species such as Pantoea stewartii (WO03/016503; WO 02/079395), Erwinia uredovora (EP 393690 B1; Misawa etal., J. Bacteriol., 172(12):6704-6712 (1990)), Erwinia herbicola (Hundleet al., Mol. Gen Genet., 245(4):406-416 (1994); Hundle et al., FEBSLett. 315(3):329-334 (1993); Schnurr et al., FEMS Microbiol. Lett.,78(2-3):157-161 (1991); and U.S. Pat. No. 5,684,238), Agrobacteriumaurantiacum (Misawa et al., J. Bacteriol., 177(22):6575-6584 (1995);U.S. Pat. No. 5,811,273), Alcaligenes sp. (U.S. Pat. No. 5,811,273),Flavobacterium sp. (U.S. Pat. No. 6,677,134; U.S. Pat. No. 6,291,204; US2002147371; WO 2004029275; and Pasamontes et al., Gene, 185(1):35-41(1997)), Paracoccus sp. (CN 1380415), Haematococcus pluvialis (WO00/061764; Linden, H., Biochimica et Biophysica Acta, 1446(3):203-212(1999)), Brevundimonas vesicularis DC263 (U.S. Ser. No. 60/601,947),Enterobacteriaceae strain DC260 (U.S. Ser. No. 10/808,979, and plantspecies such as Arabidopsis thaliana (Tian, L. and DellaPenna, D., PlantMol. Biol., 47(3):379-388 (2001); US 2002102631).

Carotenoid ketolases are enzymes that introduce keto groups to theβ-ionone ring of the cyclic carotenoids, such as β-carotene, echinenone,β-cryptoxanthin, adonixanthin, 3′-hydroxyechinenone,3-hydroxyechinenone, and zeaxanthin to produce ketocarotenoids. Examplesof ketocarotenoids include, but are not limited to astaxanthin,canthaxanthin, adonixanthin, adonirubin, echinenone,3-hydroxyechinenone, 3′-hydroxyechinenone, 4-keto-gamma-carotene,4-keto-rubixanthin, 4-keto-torulene, 3-hydroxy-4-keto-torulene,deoxyflexixanthin, and myxobactone.

Several classes of carotenoid ketolases have been reported (Hannibal etal., J. Bacteriol., 182: 3850-3853 (2000)). These include CrtW ketolasesfrom Agrobacterium aurantiacum (Misawa et al., J. Bacteriol.,177(22):6575-6584 (1995); WO 99/07867), Bradyrhizobium sp. ORS278(Hannibal et al., J. Bacteriol., 182(13):3850-3853 (2000)),Brevundimonas aurantiaca (De Souza et al., WO 02/79395), Paracoccusmarcusii (Yao et al., CN1380415); Bkt ketolases from Haematococcuspluvialis (Sun et al., Proc. Natl. Acad. Sci. USA, 95(19):11482-11488(1998); Linde, H. and Sandmann, G., EP1173579; Breitenbach et al., FEMSMicrobiol. Lett., 404(2-3):241-246 (1996)); and CrtO ketolases fromSynechocystis sp. (Lagarde et al., Appl. Environ. Microbiol.,66(1):64-72 (2000); Masamoto et al., Plant Cell Physiol., 39(5):560-564(2000); FR 2792335; Cheng et al., WO 03/012056 corresponding to U.S.Ser. No. 10/209,372)), Rhodococcus erythropolis (Cheng et al., supra),Deinococcus radiodurans (Cheng et al., supra), and Gloeobacter violaceus(Nakamura et al., DNA Res., 10:181-201 (2003)). It should be noted thatthe CrtO ketolase reported in Haematococcus pluvialis (Harker, M. andHirschberg, J., FEBS Lett., 404(2-3):129-134 (1997); U.S. Pat. No.5,965,795; U.S. Pat. No. 5,916,791; and U.S. Pat. No. 6,218,599) appearsto be a CrtW/Bkt-type ketolase based on the size (nucleotide codingsequence length <1000 bp) and homology to other CrtW/Bkt ketolases. Bktketolases appear to be closely related to CrtW ketolases, sharing verylittle structural similarity to the CrtO ketolases based on nucleotideand amino acid sequence comparisons (Cheng, et al, supra). For example,a search of the publicly available sequences using the Haematococcuspluvialis Bkt ketolase sequence returned matches that most closelymatched other CrtW-type ketolases. CrtW/Bkt ketolases are generallyencoded by nucleic acid fragments about 800-1000 bp in length, whileCrtO ketolases are normally encoded by a nucleic acid fragments of about1.6 kb in size. Cheng et al. defines CrtO ketolases based on theexistence of six conserved motifs considered diagnostic for all CrtOketolases. The reported CrtO ketolases from Rhodococcus erythropolis,Deinococcus radiodurans, and Synechocystis sp. PCC6803 are comprised ofthese diagnostic motifs (U.S. Ser. No. 10/209,372).

The wildtype CrtO ketolases reported by Cheng et al. generally exhibitmuch lower activity when producing ketocarotenoids (i.e. canthaxanthin)from β-carotene in comparison to the reported CrtW ketolases (U.S. Ser.No. 10/209,372). U.S. Ser. No. 10/209,372 reports that, the use ofrecombinatly expressed R. erythropolis AN12 CrtO ketolase resulted inonly 30% conversion of the initial substrate (β-carotene) intocanthaxanthin (35% of the initial β-carotene was converted to echinenonewith the remaining 35% remaining as β-carotene).

For biosynthesis of astaxanthin, a carotenoid ketolase and a carotenoidhydroxylase have to interact efficiently (Steiger, S. and Sandmann, G.,Biotechnol Lett., 26:813-817 (2004)). As shown in FIG. 1, manycarotenoid ketolases and carotenoid hydroxylases exhibit some level ofsubstrate flexibility. This leads to a variety of possible enzymaticreactions (producing various intermediates from β-carotene) that may benecessary to produce astaxanthin. Depending upon the activity andsubstrate specificity of both the ketolase and hydroxylase used, it isoften difficult to predict those combinations that will result inoptimal production of astaxanthin. For example, it has been reportedthat hydroxylases from cyanobacteria are not able to accept echinenoneor canthaxanthin as substrates for hydroxylation. Conversely, certainketolases have been reported to be unable to use hydroxylatedcarotenoids, such as zeaxanthin, as suitable substrates (Steiger andSandmann, supra). Coexpression of a carotenoid ketolase and a carotenoidhydroxylase that are able to efficiently work together is crucial forproducing substantial amounts of astaxathin in a recombinant host cell.When using a recombinant host cell capable of producing suitable amountsof β-carotene, one must take into account a variety of variables thatfactor into optimal astaxanthin production including, but not limitedto 1) substrate flexibility associated with each ketolase and thehydroxylase used, 2) the ability of each enzyme to efficientlyhydroxylate/ketolate one or more of the possible carotenoid substrates,and 3) the relative balance of ketolase and hydroxylase enzymaticactivity. For example, one can invision a scenario where a ketolase,which selectively uses β-carotene as a substrate, should not becoexpressed with a hydroxylase having much higher activity forβ-carotene. In such an instance, the majority of the β-carotene would beexpected to be converted into hydroxylated carotenoids (such aszeaxanthin) that may not be recognized by the ketolase, resulting inless than optimal production of astaxanthin.

The CrtO ketolase from R. erythropolis AN12 has been protein engineeredfor increased canthaxanthin production (U.S. Ser. No. 60/577,970).Coexpression of the CrtO ketolase with a CrtZ cartenoid hydroxylase wasexpected to result in the production of astaxanthin (FIG. 1). However,as described in the present disclosure, coexpression of the bestcanthaxanthin producing CrtO ketolase mutant (“320SHU001” hereinreferred to as “crtO-SHU0001”) from U.S. Ser. No. 60/577,970 withseveral different CrtZ hydroxylases did not result in the expectedproduction of astaxanthin.

The problem to be solved is to provide nucleic acid molecules encodingat least one CrtO ketolase and at least one CrtZ hydroxylase that canefficiently work together to produce astaxanthin in a recombinant hostcell. A further problem to be solved is to provide a method to producematched pairs of carotenoid ketolases and carotenoid hydroxylases havingastaxanthin biosynthesis activity.

The stated problem has been solved by providing several CrtOketolase/CrtZ hydroxylase mutants exhibiting improved astaxanthinproduction in the context of a carotenoid biosynthetic pathway. Anucleic acid fragment (comprised of crtOZ genes) encoding enzymesincapable of producing more than trace amounts astaxanthin was proteinengineered using errror-prone PCR to create several CrtO/Z combinationshaving the ability to produce significant amounts of astaxanthin whenexpressed in a recombinant host cell. Methods to produce and/or alterastaxanthin production in recombinant host cells using the present genesare also provided.

Additionally, a method to produce combinations of carotenoid ketolasesand carotenoid hydroxylases having improved astaxanthin biosynthesisactivity is also provided. The method is comprised of simultaneouslymutating nucleic acid fragments encoding one or more carotenoidketolases and one or more carotenoid hydroxylases and screeningrecombinants for improvements in astaxanthin production.

SUMMARY OF THE INVENTION

The present invention provides nucleic acid molecules encoding CrtOketolases and CrtZ hydroxylases useful for producing astaxanthin inrecombinant host cells engineered to produce β-carotene. A crtOZ genecombination encoding enzymes incapable of producing significant amountsof astaxanthin was simultaneously mutated using error-prone PCR,creating several mutant CrtO/Z enzyme pairs capable of producingincreased amounts of astaxanthin.

Accordingly the invention provides an isolated nucleic acid moleculeencoding at least one carotenoid ketolase and at least one carotenoidhydroxylase, said nucleic acid molecule comprising:

-   -   a) a nucleic acid fragment encoding a carotenoid ketolase having        an amino acid sequence selected from the group consisting of SEQ        ID NO: 17 and SEQ ID NO: 22; and    -   b) a isolated nucleic acid fragment encoding a carotenoid        hydroxylase having an amino acid sequence selected from the        group consisting of SEQ ID NO: 19 and SEQ ID NO: 24: or        an isolated nucleic acid molecule completely complementary to        the nucleic acid molecule comprising the elements of (a) and        (b).

In similar fashion the invention provides an isolated nucleic acidmolecule encoding at least one carotenoid ketolase and at least onecarotenoid hydroxylase, said nucleic acid molecule comprising:

-   -   a) a nucleic acid fragment encoding a carotenoid ketolase having        a nucleic acid sequence selected from the group consisting of        SEQ ID NO: 16 and SEQ ID NO: 21; and    -   b) a nucleic acid fragment encoding a carotenoid hydroxylase        having a nucleic acid sequence selected from the group        consisting of SEQ ID NO: 18 and SEQ ID NO: 23; or        an isolated nucleic acid molecule completely complementary to        the nucleic acid molecule comprising the elements of (a) and        (b).

Alternatively the invention provides an isolated nucleic acid moleculeencoding a carotenoid ketolase and a carotenoid hydroxylase, saidisolated nucleic acid molecule comprising:

-   -   a) a nucleic acid fragment encoding a carotenoid ketolase having        an amino acid sequence as represented by SEQ ID NO: 17 and    -   b) a nucleic acid fragment encoding a carotenoid hydroxylase        enzyme having an amino acid sequence as represented by SEQ ID        NO: 19; or        an isolated nucleic acid molecule completely complementary to        the nucleic acid molecule comprising the elements of (a) and        (b).

In another embodiment the invention provides an isolated nucleic acidmolecule encoding a carotenoid ketolase and a carotenoid hydroxylase,said isolated nucleic acid molecule comprising:

-   -   a) a nucleic acid fragment encoding a carotenoid ketolase having        an amino acid sequence as represented by SEQ ID NO: 21; and    -   b) a nucleic acid fragment encoding a carotenoid hydroxylase        enzyme having an amino acid sequence as represented by SEQ ID        NO: 23; or        an isolated nucleic acid molecule completely complementary to        the nucleic acid molecule comprising the elements of (a) and        (b).

In other embodiment the invention provides polypeptides encoded by theinstant sequences, genetic chimera of the instant sequences, and hostcells transformed with the same.

In one embodiment the invention provides a method for the production ofastaxanthin comprising:

-   -   a) providing a transformed host cell that produces β-carotene        and which comprises the chimeric gene cluster of the invention        encoding at least one carotenoid ketolase enzyme and at least        one carotenoid hydroxylase enzyme; and    -   b) growing the transformed host cell of (a) under suitable        conditions whereby astaxanthin is produced.

Similarly the invention provides a method of altering astaxanthinbiosynthesis in an organism comprising:

-   -   a) providing a host cell capable of producing astaxanthin;    -   b) introducing into said host cell the nucleic acid molecule of        the invention; wherein said nucleic acid molecule encodes a        carotenoid ketolase gene and a carotenoid hydroxylase gene; and    -   c) growing the host cell of (b) under conditions whereby the        nucleic acid molecule is expressed and astaxanthin biosynthesis        is altered.

In an other embodiment the invention provides a method to generate andidentify nucleic acid molecules encoding a carotenoid ketolase and acarotenoid hydroxylase having improved astaxanthin biosynthesis activitycomprising:

-   -   a) providing a host cell capable of producing β-carotene;    -   b) providing a starting pair of genes comprising a carotenoid        ketolase gene and a carotenoid hydroxylase;    -   c) exposing said starting pair of genes simultaneously to        mutational conditions in vitro to form a mutated gene pair;        wherein at least one nucleotide has been altered in either one        or both of said carotenoid ketolase gene or said carotenoid        hydroxylase gene;    -   d) operably linking the mutated gene pair to a suitable        regulatory sequence;    -   e) transforming the host cell of step a) with the mutated gene        pair from step d) to produce a recombinant host cell;    -   f) growing the recombinant host cell under conditions whereby        astaxanthin is produced;    -   g) measuring the amount of astaxanthin produced in step f) and        selecting those transformants having increased astaxanthin        production relative to the level of astaxanthin produced by the        starting pair of genes in the host cell; and    -   h) identifying the mutated gene pair from the selected        transformants which have increased astaxanthin biosynthesis        activity.

BRIEF DESCRIPTION OF THE FIGURES BIOLOGICAL DEPOSITS AND SEQUENCEDESCRIPTIONS

The invention can be more fully understood from the following figure,detailed description, biological deposits, and the accompanying sequencedescriptions, which form a part of this application.

FIG. 1 shows common carotenoid products produced by carotenoid ketolasesin conjunction with carotenoid hydroxylase enzymes using β-carotene asthe substrate.

The following sequences comply with 37 C.F.R. 1.821-1.825 (“Requirementsfor Patent Applications Containing Nucleotide Sequences and/or AminoAcid Sequence Disclosures—the Sequence Rules”) and are consistent withWorld Intellectual Property Organization (WIPO) Standard ST.25 (1998)and the sequence listing requirements of the EPO and PCT (Rules 5.2 and49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions). The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the nucleotide sequence of a mutant crtO carotenoidketolase coding sequence, designated as “crtO-SHU001”, previouslycreated by protein engineering the wild type crtO gene from Rhodococcuserythropolis AN12 for improved ketocarotenoid production (U.S. Ser. No.60/577,970).

SEQ ID NO: 2 is deduced amino acid sequence of crtO-SHU001.

SEQ ID NO: 3 is the nucleotide sequence of primer crtO-SHU001-F.

SEQ ID NO: 4 is the nucleotide sequence of primer crtO-SHU001-R.

SEQ ID NO: 5 is the nucleotide sequence of the crtZ hydroxylase codingsequence isolated from Brevundimonas vesicularis DC263 (U.S. Ser. No.60/601,947).

SEQ ID NO: 6 is the deduced amino acid sequence of the CrtZ hydroxylasefrom Brevundimonas vesicularis DC263 (U.S. Ser. No. 60/601,947).

SEQ ID NO: 7 is the nucleotide sequence of primer crtZ-263_F2.

SEQ ID NO: 8 is the nucleotide sequence of primer crtZ-263_R2.

SEQ ID NO: 9 is the nucleotide sequence of the crtZ hydroxylase codingsequence isolated from Enterobacteriaceae DC260 (U.S. Ser. No.10/808,979).

SEQ ID NO: 10 is the deduced amino acid sequence of the CrtZ hydroxylaseisolated from Enterobacteriaceae DC260 (U.S. Ser. No. 10/808,979).

SEQ ID NO: 11 is the nucleotide sequence of primer crtZ-DC260-F.

SEQ ID NO: 12 is the nucleotide sequence of primer crtZ-DC260-R.

SEQ ID NO: 13 is the nucleotide sequence of primer 334F1.

SEQ ID NO: 14 is the nucleotide sequence of primer 334R1.

SEQ ID NO: 15 is the nucleotide sequence of the nucleic acid fragmentcomprised of the mutant crtOZ coding sequences found in plasmidpDCQ356M4003.

SEQ ID NO: 16 is the nucleotide sequence of the crtO ketolase codingsequence found in plasmid pDCQ356M4003.

SEQ ID NO: 17 is the deduced amino acid sequence of the mutant CrtOketolase from plasmid pDCQ356M4003.

SEQ ID NO: 18 is the nucleotide sequence of the crtZ hydroxylase codingsequence found in plasmid pDCQ356M4003.

SEQ ID NO: 19 is the deduced amino acid sequence of the mutant CrtZhydroxylase from plasmid pDCQ356M4003.

SEQ ID NO: 20 is the nucleotide sequence of the nucleic acid fragmentcomprised of the mutant crtOZ coding sequence from plasmid pDCQ356M4005.

SEQ ID NO: 21 is the nucleotide sequence of the mutant crtO ketolasecoding sequence found in plasmid pDCQ356M4005.

SEQ ID NO: 22 is the deduced amino acid sequence of the mutant CrtOketolase from plasmid pDCQ356M4005.

SEQ ID NO: 23 is the nucleotide sequence of the mutant crtZ hydroxylasecoding sequence from plasmid pDCQ356M4005.

SEQ ID NO: 24 is the deduced amino acid sequence of the mutant CrtZhydroxylase from plasmid pDCQ356M4005.

BRIEF DESCRIPTION OF THE BIOLOGICAL DEPOSIT

The following biological deposit has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the purposes of Patent Procedure: DepositorIdentification Int'l. Depository Reference Designation Date of DepositMethylomonas 16a ATCC# PTA-2402 Aug. 22, 2000

As used herein, “ATCC” refers to the American Type Culture CollectionInternational Depository Authority located at ATCC, 10801 UniversityBlvd., Manassas, Va. 20110-2209, USA. The “International DepositoryDesignation” is the accession number to the culture on deposit withATCC.

The listed deposit will be maintained in the indicated internationaldepository for at least thirty (30) years and will be made available tothe public upon the grant of a patent disclosing it. The availability ofa deposit does not constitute a license to practice the subjectinvention in derogation of patent rights granted by government action.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to nucleic acid molecules encoding CrtO ketolasesand CrtZ hydroxylases useful for astaxanthin production. Coexpression ofthe present crtOZ genes in a recombinant host cell resulted in asignificant increase in astaxanthin production. In another embodiment,methods to produce and/or alter astaxanthin production in a recombinanthost cell using the present crtOZ genes are also provided. In yet afurther embodiment, a method to produce matched carotenoid ketolase andcarotenoid hydroxylase having an improvement in astaxanthin productionis also provided.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

As used herein, the terms an “isolated nucleic acid fragment” and “anisolated nucleic acid molecule” will be used interchangeably and willmean a polymer of RNA or DNA that is single- or double-stranded,optionally containing synthetic, non-natural or altered nucleotidebases. An isolated nucleic acid fragment in the form of a polymer of DNAmay be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA.

The term “isoprenoid” or “terpenoid” refers to the compounds are anymolecule derived from the isoprenoid pathway including 10 carbonterpenoids and their derivatives, such as carotenoids and xanthophylls.

The term “carotenoid” refers to a compound composed of a polyenebackbone which is condensed from five-carbon isoprene unit. Carotenoidscan be acyclic or terminated with one (monocyclic) or two (bicyclic)cyclic end groups. The term “carotenoid” may include both carotenes andxanthophylls. A “carotene” refers to a hydrocarbon carotenoid. Carotenederivatives that contain one or more oxygen atoms, in the form ofhydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functionalgroups, or within glycosides, glycoside esters, or sulfates, arecollectively known as “xanthophylls”. Carotenoids that are particularlysuitable in the present invention are monocyclic and bicycliccarotenoids.

The term “carotenoid ketolase” or “ketolase” refers to an enzyme thatcan add keto groups to the ionone ring of either monocyclic or bicycliccarotenoids. Two distinct classes of carotenoid ketolase have beenreported. The first class will be referred to as the CrtW/Bkt-typeketolase and are generally encoded by a nucleotide sequence ofapproximately 800-1000 bp in length. The second class of ketolase is theCrtO-type ketolase. The CrtO-type ketolase is normally encoded bynucleotide sequence of approximately 1.6 kb in length and exhibits nostructural similarity to the CrtW/Bkt ketolases (See U.S. Ser. No.60/577,970).

The terms “crtO-SHU001” and “320SHU001” will be used interchangeably andrefer to the nucleic acid fragment encoding a mutant CrtO ketolasepreviously engineered for increased ketocarotenoid (i.e. canthaxanthin)production (U.S. Ser. No. 60/577,970; hereby incorporated by reference).The crtO-SHU001 gene (SEQ ID NO: 1) was used as the starting gene forerror-prone PCR reactions to create crtOZ combinations having theability to produce significant amounts of astaxanthin.

The term “carotenoid hydroxylase” or “hydroxylase” or “CrtZ hydroxylase”refers to an enzyme that adds hydroxyl groups to the ionone ring ofeither monocyclic or bicylic carotenoids. Two CrtZ hydroxylases (crtZfrom Brevundimonas vesicularis DC263; U.S. Ser. No. 60/601,947 and crtZfrom Enterobacteriaceae DC260; U.S. Ser. No. 10/808,979; bothincorporated herein by reference) were individually coexpressed with aCrtO ketolase (crtO-SHU001) to determine if either crtOZ combinationcould produce astaxanthin in E. coli or Methylomonas 16a. Neither of theCrtZ hydroxylases coexpressed with crtO-SHU001 could produce astaxanthinin significant amounts. The CrtO ketolase gene crtO-SHU001 and the crtZgene (SEQ ID NO: 5) from Brevundimonas vesicularis DC263 were selectedand used as the starting genes for simultaneous error-prone PCRexperiments used to create crtOZ combinations (SEQ ID NOs: 15 and 20)having the ability to produce significant amounts of astaxanthin.

The term “crtO/Z” will refer to an isolated nucleic acid moleculeencoding polypeptide(s) having carotenoid hydroxylase and carotenoidketolase activity as defined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable to hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid fragments that are complementary tothe complete sequences as reported in the accompanying Sequence Listing.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., andGriffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis inMolecular Biology (von Heinje, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) StocktonPress, NY (1991). Preferred methods to determine identity are designedto give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences was performed using the Clustal method ofalignment (Higgins and Sharp, CABIOS, 5:151-153 (1989)) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the instant microbialpolypeptides as set forth in SEQ ID NOs: 19, 22, and 24 The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

As used herein, “conservative substitution” is used to describealterations in a gene which result in the production of a chemicallyequivalent amino acid at a given site, but do not effect the functionalproperties of the encoded protein are common. For the purposes of thepresent invention, substitutions are defined as exchanges within one ofthe following five groups:

-   -   1. Small aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr (Pro, Gly);    -   2. Polar, negatively charged residues and their amides: Asp,        Asn, Glu, Gln;    -   3. Polar, positively charged residues: His, Arg, Lys;    -   4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys);        and    -   5. Large aromatic residues: Phe, Tyr, Trp.        Thus, a codon for the amino acid alanine, a hydrophobic amino        acid, may be substituted by a codon encoding another less        hydrophobic residue (such as glycine) or a more hydrophobic        residue (such as valine, leucine, or isoleucine). Similarly,        changes which result in substitution of one negatively charged        residue for another (such as aspartic acid for glutamic acid) or        one positively charged residue for another (such as lysine for        arginine) can also be expected to produce a functionally        equivalent product. In many cases, nucleotide changes which        result in alteration of the N-terminal and C-terminal portions        of the protein molecule would also not be expected to alter the        activity of the protein. In another embodiment, the present        nucleic acid fragments may optionally include those having        conservative substitutions.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available. For example, the preferredcodon usage for Methylomonas sp. 16a (ATCC PTA-2402) has been previouslyreported in U.S. Ser. No. 60/527,083; hereby incorporated by reference.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The “3′non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal (normallylimited to eurkaryotes) is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO99/28508). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that is not translated yet hasan effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. As used herein, the host cell genome includes bothchromosomal or extrachromosomal (i.e. a vector) genes with the hostcell. Host organisms containing the transformed nucleic acid fragmentsare referred to as “transgenic” or “recombinant” or “transformed”organisms.

The terms “Rhodococcus erythropolis AN12”, “Rhodococcus erythropolisstrain AN12” or “AN12” will be used interchangeably and refer to theRhodococcus erythropolis AN12 strain (U.S. Ser. No. 10/209,372).

The terms “Brevundimonas vesicularis” and “Brevunidmonas vesicularisstrain DC263” will be used interchangeably and refer to theBrevundimonas vesicularis DC263 strain (U.S. Ser. No. 60/601,947).

The term “Enterobacteriaceae DC260” or “DC260” will be usedinterchangeably and refer to the Enterobacteriaceae DC260 strain (U.S.Ser. No. 10/808,979).

The term “pDCQ334” refers to a plasmid comprised of the crtWZEidiYIBgene cluster capable of producing astaxanthin in a recombinant host cell(U.S. Ser. No. 60/527,083). The plasmid was created by cloning a codonoptimized version (optimized for Methylomonas sp. 16a) of theAgrobacterium aurantiacum crtWZ genes (U.S. Pat. No. 5,972,690; GenBank®D58420) upstream of crtE in the native crtEidiYIB cluster from P.agglomerans DC404 (U.S. Ser. No. 10/808,807) to form the operoncrtWZEidiYIB (U.S. Ser. No. 60/527,083). The crtWZEidiYIB genes wereorganized in an operon and were under the control of the chloramphenicolresistant gene promoter of the parent vector (broad host range vectorpBHR1; MoBiTec, LLC, Marco Island, Fla.).

The term “pDCQ353” refers to the plasmid comprised of the crtO-SHU001gene cloned into a pTrcHis2-TOPO vector (Invitrogen, Carlsbad, Calif.).

The term “pDCQ354” refers the plasmid created by replacing the crtWZgene cluster in pDCQ334 with a nucleic acid fragment comprised of thecrtO-SHU001 gene, resulting in the operon crtOEidiYIB. Recombinant cellscontaining pDCQ354 produce canthaxanthin.

The term “pDCQ352” refers to the plasmid created by cloning the crtZgene from Brevunidmonas vesicularis strain DC263 into a pTrcHis2-TOPOvector.

The term “pDCQ355” refers to the plasmid created by cloning the crtZgene from Enterobacteriaceae DC260 into a pTrcHis2-TOPO vector.

The term “pDCQ356” refers to the plasmid created by cloning the crtZgene from Brevunidmonas vesicularis strain DC263 into the SpeIrestriction site of pDCQ354, resutling in the operon crtOZEidiYIB.Transformants harboring pDCQ356 were unable to produce significantamounts of astaxanthin. Plasmid pDCQ356 was selected as the “control”plasmid used to evaluate various mutant crtOZ gene clusters for theirability to produce astaxanthin. The crtOZ gene cluster from pDCQ356 wasused as a template for error-prone PCR.

The term “pDCQ357” refers to the plasmid created by cloning the crtZgene from Enterobacteriaceae DC260 into the SpeI restriction site ofpDCQ354, resulting in the operon crtOZEidiYIB. Transformants harboringpDCQ357 were unable to produce significant amounts of astaxanthin.

The term “pDCQ356M4003” refers to a plasmid created by removing thecrtOZ insert from pDCQ356 and inserting a mutagenized crtOZ genecluster. The mutant CrtO was comprised on an amino acid sequence asshown in SEQ ID NO: 17. The mutant CrtZ was comprised of an amino acidsequence as shown in SEQ ID NO: 19. Transformants harboring pDCQ356M4003were able to produce significant amounts of astaxanthin.

The term “pDCQ356M4005” refers to a plasmid created by removing thecrtOZ insert from pDCQ356 and inserting a mutagenized crtOZ genecluster. The mutant CrtO was comprised on an amino acid sequence asshown in SEQ ID NO: 22. The mutant CrtZ was comprised of an amino acidsequence as shown in SEQ ID NO: 24. Transformants harboring pDCQ356M4005were able to produce significant amounts of astaxanthin.

The term “carbon substrate” refers to a carbon source capable of beingmetabolized by host organisms of the present invention and particularlycarbon sources selected from the group consisting of monosaccharides,disaccharides, polysaccharides, and one-carbon substrates or mixturesthereof. In one embodiment, the carbon substrate is a single carbonsubstrate selected from the group consisting of methane and/or methanol.

The term “Entner-Douderoff pathway” refers to a series of biochemicalreactions for conversion of hexoses such as glucose or fructose to theimportant 3-carbon cellular intermediates pyruvate and glyceraldehyde3-phosphate without any net production of biochemically useful energy.The key enzymes unique to the Entner-Douderoff pathway are the6-phosphogluconate dehydratase and a ketodeoxyphospho-gluconatealdolase.

The term “Embden-Meyerhof pathway” refers to the series of biochemicalreactions for conversion of hexoses such as glucose and fructose toimportant cellular 3-carbon intermediates such as glyceraldehyde 3phosphate, dihydroxyacetone phosphate, phosphoenol pyruvate andpyruvate. These reactions typically proceed with net yield ofbiochemically useful energy in the form of ATP. The key enzymes uniqueto the Embden-Meyerhof pathway are the phosphofructokinase and fructose1,6 bisphosphate aldolase.

The term “C₁ carbon substrate” or “single carbon substrate” refers toany carbon-containing molecule that lacks a carbon-carbon bond. Examplesare methane, methanol, formaldehyde, formic acid, formate, methylatedamines (e.g., mono-, di-, and tri-methyl amine), methylated thiols, andcarbon dioxide. In another embodiment, the C₁ carbon substrate ismethanol and/or methane.

The term “C₁ metabolizer” refers to a microorganism that has the abilityto use a single carbon substrate as its sole source of energy andbiomass. C₁ metabolizers will typically be methylotrophs and/ormethanotrophs.

The term “methylotroph” means an organism capable of oxidizing organiccompounds that do not contain carbon-carbon bonds. Where themethylotroph is able to oxidize CH₄, the methylotroph is also amethanotroph. In another embodiment, the methylotroph is capable ofusing methanol and/or methane as a carbon source.

The term “methanotroph” or “methanotrophic bacteria” means a prokaryotecapable of utilizing methane as its primary source of carbon and energy.Complete oxidation of methane to carbon dioxide occurs by aerobicdegradation pathways. Typical examples of methanotrophs useful in thepresent invention include (but are not limited to) the generaMethylomonas, Methylobacter, Methylococcus, and Methylosinus.

The term “high growth methanotrophic bacterial strain” refers to abacterium capable of growth with methane or methanol as the sole carbonand energy source and which possesses a functional Embden-Meyerof carbonflux pathway resulting in a high rate of growth and yield of cell massper gram of C₁ substrate metabolized. The specific “high growthmethanotrophic bacterial strain” described herein is referred to as“Methylomonas 16a”, “16a” or “Methylomonas sp. 16a”, which terms areused interchangeably and which refer to the Methylomonas sp. 16a (ATCCPTA-2402) strain (U.S. Pat. No. 6,689,601) and derivatives thereof whichpossess a functional Embden-Meyerof carbon flux pathway.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction.“Transformation cassette” refers to a specific vector containing aforeign gene and having elements in addition to the foreign gene thatfacilitates transformation of a particular host cell. “Expressioncassette” refers to a specific vector containing a foreign gene andhaving elements in addition to the foreign gene that allow for enhancedexpression of that gene in a foreign host.

The term “altered biological activity” will refer to an activity,associated with a protein encoded by a microbial nucleotide sequencewhich can be measured by an assay method, where that activity is eithergreater than or less than the activity associated with the nativemicrobial sequence. “Enhanced biological activity” refers to an alteredactivity that is greater than that associated with the native sequence.“Diminished biological activity” is an altered activity that is lessthan that associated with the native sequence.

In the present application, matched pairs of protein engineered CrtOketolases and CrtZ hydroxylases are provided that have improved“astaxanthin biosynthesis activity” when compared the genes from whichthey were developed. In the present application the crtO gene was themutant “crtO-SHU0001” and was previously engineered for increasedketocaroenoid production; see U.S. Ser. No. 60/577,970). “Astaxanthinbiosynthesis activity” refers to the amount of astaxanthin producedunder specified conditions in a recombinant host cell. Improvedastaxanthin biosynthesis activity is determined by comparing theactivity of mutant genes expressed in a host cell and function in thecontext of an existing or engineered carotenoid enzymatic pathway withthe expression of the genes from which they were derived functioning inthe same host cell under the same conditions. Thus in the presentapplication increases in astaxanthin biosynthesis activity were measuredby comparing the amount of astaxanthin produced by mutant crtO/Z gene(s)versus the astaxanthin biosynthesis activity of host cells expressingthe crtOZ genes from plasmid pDCQ356 (original genes) under identicalreaction conditions. The matched pair of enzymes (CrtO and CrtZ)expressed from pDCQ356 (i.e. the “starting genes”) exhibitedinsignificant astaxanthin production when expressed in a recombinanthost cell capable of producing β-carotene. The expression system used toevaluate each mutant and the corresponding control was identical. Therecombinant protein expression level of each mutant crtOZ pair wasessentially identical. Improvements in the percentage yield ofastaxanthin production were attributed to structural differencesassociated with the CrtO ketolase and/or CrtZ hydroxylase. The crtOZcoding sequences were simulatenous mutated using error-prone PCR toproduce a matched gene pair that optimally produces astaxanthin. Inanother embodiment, the present mutant crtO genes or crtZ genes may beindividually matched and recombinantly expressed with other carotenoidketolases and/or hydroxylases for the production of astaxanthin. Thestructural differences are represented by the nucleotide and amino acidsequences provided herein.

The term “mutational conditions” refers to conditions that result ingene mutations. Typical mutational conditions will include theconditions prescribed by error Prone PCR ((Melnikov et al., NucleicAcids Research, 27(4):1056-1062 (1999); Leung et al., Techniques,1:11-15 (1989); and Zhou et al., Nucleic Acids Res., 19:6052-6052(1991)); and methods of gene schuffling (see for example U.S. Pat. No.5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721; and U.S.Pat. No. 5,837,458; and Tang et al., U.S. Ser. No. 10/374,366; andIkeuchi et al., Biotechnol. Prog., 2003, 19 1460-7).

“Amplification” is the process in which replication is repeated incyclic manner such that the number of copies of the “template nucleicacid” is increased in either a linear or logarithmic fashion.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) theFASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.); and 5.)the Vector NTI version 7.0 programs (Informax, Inc., Bethesda, Md.).Within the context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters (set by the manufacturer)which originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

The present invention provides several mutant crtOZ genes having theability to produce significant amounts of astaxanthin when compared tothe astaxanthin synthesizing activity of the “starting genes”(exemplified herein using the crtO-SHU001 gene (SEQ ID NO: 1)coexpressed with the crtZ gene (SEQ ID NO: 5) from Brevundimonasvesicularis DC263). Improvements in astaxanthin synthesis were conductedin recombinant hosts engineered to produce suitable amounts ofβ-carotene. Improvements in astaxanthin synthesis were determined bycomparing the percentage yield of astaxanthin in the various mutantsproduced to the activity of the “starting genes”.

In one embodiment, paired CrtO/Z enzymes of the present invention arethose having an increase in the percentage yield of astaxanthin of atleast 5% when compared to the percentage yield of astaxanthin inrecombinant hosts coexpressing the crtO-SHU001 ketolase gene and thecrtZ hydroxylase gene from Brevundimonas vesicularis DC263 underidentical reaction conditions. More preferred CrtO/Z enzyme pairs of thepresent invention are those exhibiting at least a 10% increase in thepercentage yield of astaxanthin when coexpressed in a recombinant hostcell. Even more preferred CrtO/Z enzyme pairs are those exhibiting at a20% increase in the percentage yield of astaxanthin. Comparisons inastaxanthin synthesizing activity can be conducted under a variety ofreaction conditions depending upon the selected host organism. Suitablecomparisons are those conducted between the engineered crtOZ gene pairof interest and a suitable control (i.e. another crtOZ gene cluster)recombinantly expressed (using identical expression systems) underidentical reaction conditions (i.e. recombinant host cells capable ofproducing suitable levels of β-carotene). In a further embodiment,“significant astaxanthin production” will be used to describedrecombinant production of astaxanthin where at least about 3% of thetotal carotenoids produced is astaxanthin, preferably at least about 5%,more preferably at least about 15%, and most preferably at least about20%.

In one embodiment, a method to produce matched pairs of carotenoidketolase(s) and carotenoid hydroxylase(s) for increased or optimizedproduction of astaxanthin is also provided.

In another embodiment, the process to produced carotenoid ketolases andcarotenoid hydroxylases having an improved ability to produceastaxanthin is not limited to CrtO-type carotenoid ketolases or CrtZcarotenoid hydroxylases. The method comprises 1) providing a recombinanthost cell capable of producing β-carotene, 2) providing at least onecarotenoid ketolase and at least one carotenoid hydroxylase (i.e.“starting genes”), 3) simulatenously treating the genes encoding atleast one carotenoid ketolase and at least one carotenoid hydroxylaseusing a mutagenizing process or under mutational conditions, 4)transforming the recombinant host cell with the mutated genes, 5)screening the recombinant host cell for increased astaxanthin productionrelative to recombinant host cells expressing the starting genes, and 6)selecting those recombinant hosts with increased astaxanthin production,and optionally isolating the mutated gene pair and optionally repeatingsteps 1) through step 6).

The present CrtO/Z ketolase/hydroxylase pairs may be used in vitro or invivo in for the production of astaxanthin from β-carotene and/orintermediates in the synthesis of astaxanthin as shown in FIG. 1.

Recombinant Expression of crtO/Z—Microbial

The genes and gene products of the instant sequences may be used inheterologous host cells, particularly in the cells of microbial hosts,for the production of carotenoid compounds and particularly for theproduction of astaxanthin.

Preferred heterologous host cells for expression of the present mutantcrtO/Z genes are microbial hosts that can be found broadly within thefungal or bacterial families and which grow over a wide range oftemperature, pH values, and solvent tolerances. For example, it iscontemplated that any of bacteria, yeast, and filamentous fungi will besuitable hosts for expression of the present nucleic acid fragments.Because of transcription, translation and the protein biosyntheticapparatus is the same irrespective of the cellular feedstock, functionalgenes are expressed irrespective of carbon feedstock used to generatecellular biomass. Large-scale microbial growth and functional geneexpression may utilize a wide range of simple or complex carbohydrates,organic acids and alcohols, saturated hydrocarbons such as methane orcarbon dioxide in the case of photosynthetic or chemoautotrophic hosts.However, the functional genes may be regulated, repressed or depressedby specific growth conditions, which may include the form and amount ofnitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrientincluding small inorganic ions. In addition, the regulation offunctional genes may be achieved by the presence or absence of specificregulatory molecules that are added to the culture and are not typicallyconsidered nutrient or energy sources. Growth rate may also be animportant regulatory factor in gene expression.

Examples of potential host strains include, but are not limited tobacterial, fungal or yeast genera such as Aspergillus, Trichoderma,Saccharomyces, Pichia, Phaffia, Candida, Hansenula, or bacterial speciessuch as Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium,Erythrobacter Chlorobium, Chromatium, Flavobacterium, Cytophaga,Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria,Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas,Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus,Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis,Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, andMyxococcus. In one embodiment, the host strain is a methylotroph grownon methanol and/or methane. Preferred bacterial species includeEscherichia coli, Methylomonas sp. 16a, and derivatives thereof.

Microbial expression systems and expression vectors containingregulatory sequences that direct high-level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for expression of present CrtOketolases and/or CrtZ hydroxylases. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the present enzymes.

Accordingly, it is expected that introduction of chimeric genes encodingthe present bacterial enzymes under the control of the appropriatepromoters will demonstrate increased or altered astaxanthin production.It is also contemplated that it will be useful to express the instantgenes both in natural (“native”) host cells as well as heterologoushosts. Since the combination of a crtO ketolase and a crtZ hydroxylaseare typically not coexpressed together in a single native host cell, theterm “natural host cell” can be optionally defined to be any host cellwhere either one of the two genes is naturally expressed. Introductionof the present crtO/Z genes into native host cell will result in alteredlevels of existing astaxanthin production. Additionally, the instantgenes may also be introduced into non-native host bacteria where theexisting carotenoid pathway may be manipulated.

The present crtO/Z gene clusters were selected for optimal production ofastaxanthin. In another embodiment, the present genes may optionally beused to produce a variety of other carotenoids including, but are notlimited to canthaxanthin, adonixanthin, adonirubin, echinenone,3-hydroxyechinenone, 3′-hydroxyechinenone, 4-keto-gamma-carotene,4-keto-rubixanthin, 4-keto-torulene, 3-hydroxy-4-keto-torulene,deoxyflexixanthin, and myxobactone. The specific substrates for thepresent CrtO/Z enzymes are carotenoids having at least one β-iononering. Cyclic carotenoids are well known in the art and availablecommercially. In another embodiment, the cyclic carotenoid substratesinclude, but are not limited to, β-carotene, γ-carotene, zeaxanthin,rubixanthin, echinenone, and torulene. In one embodiment, the presentcrtOZ gene clusters are used to produce astaxanthin from β-carotene. Ina further embodiment, the present crtOZ gene clusters are used toproduce astaxanthin in a recombinant host cell engineered to produceβ-carotene. Expression of β-carotene synthesis genes in recombinant hostcells is well known in the art.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene or gene cluster, a selectable marker, and sequencesallowing autonomous replication or chromosomal integration. Suitablevectors comprise a region 5′ of the gene or gene cluster which harborstranscriptional initiation controls and a region 3′ of the DNA fragmentwhich controls transcriptional termination. It is most preferred whenboth control regions are derived from genes homologous to thetransformed host cell, although it is to be understood that such controlregions need not be derived from the genes native to the specificspecies chosen as a production host.

Initiation control regions or promoters which are useful to driveexpression of the present coding sequences in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these coding sequences is suitable for thepresent invention including, but not limited to: CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (e.g.,useful for expression in Saccharomyces); AOX1 (e.g., useful forexpression in Pichia); and lac, ara, tet, trp, IP_(L), IP_(R), T7, tac,and trc (e.g., useful for expression in Escherichia coli) as well as theamy, apr, npr promoters and various phage promoters useful forexpression in, e.g., Bacillus. Additionally, the deoxy-xylulosephosphate synthase or methanol dehydrogenase operon promoter (Springeret al., FEMS Microbiol Lett 160:119-124 (1998)), the promoter forpolyhydroxyalkanoic acid synthesis (Foellner et al., Appl. Microbiol.Biotechnol. 40:284-291 (1993)), promoters identified from nativeplasmids in methylotrophs (EP 296484), Plac (Toyama et al., Microbiology143:595-602 (1997); EP 62971), Ptrc (Brosius et al., Gene 27:161-172(1984)), promoters identified from methanotrophs (PCT/US03/33698), andpromoters associated with antibiotic resistance [e.g., kanamycin(Springer et al., supra; Ueda et al., Appl. Environ. Microbiol.57:924-926 (1991)), chloramphenicol, or tetracycline (U.S. Pat. No.4,824,786)] are suitable for expression in C1 metabolizers.

It may be necessary to include an artificial ribosomal binding site(“RBS”) upstream of the gene(s) to be expressed, when the RBS is notprovided by the vector. This is frequently required for the second,third, etc. gene(s) of an operon to be expressed, when a single promoteris driving the expression of a first, second, third, etc. group ofgenes. Methodology to determine the preferred sequence of a RBS in aparticular host organism will be familiar to one of skill in the art, asare means for creation of this synthetic site.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included.

Merely inserting a gene or gene cluster into a cloning vector does notensure that it will be successfully expressed at the level needed. Inresponse to the need for a high expression rate, many specializedexpression vectors have been created by manipulating a number ofdifferent genetic elements that control aspects of transcription,translation, protein stability, oxygen limitation, and secretion fromthe host cell. More specifically, the molecular features that have beenmanipulated to control gene expression include: 1.) the nature of therelevant transcriptional promoter and terminator sequences; 2.) thestrength of the ribosome binding site; 3.) the number of copies of thecloned gene(s) and whether the gene(s) are plasmid-borne or integratedinto the genome of the host cell; 4.) the final cellular location of thesynthesized foreign protein(s); 5.) the efficiency of translation in thehost organism; 6.) the intrinsic stability of the cloned gene protein(s)within the host cell; and 7.) the codon usage within the cloned gene(s),such that its frequency approaches the frequency of preferred codonusage of the host cell. Each of these types of modifications areencompassed in the present invention, as means to further optimizeexpression of the present crtOZ genes.

Finally, to promote accumulation of astaxanthin, it may be necessary toreduce or eliminate the expression of certain genes in the targetpathway or in competing pathways that may serve as sinks for energy orcarbon. Alternatively, it may be useful to over-express various genesupstream of desired carotenoid intermediates to enhance production.Methods of manipulating genetic pathways for the purposes describedabove are common and well known in the art.

For example, once a key genetic pathway has been identified andsequenced, specific genes may be up-regulated to increase the output ofthe pathway. For example, additional copies of the targeted genes may beintroduced into the host cell on multicopy plasmids such as pBR322.Alternatively the target genes may be modified so as to be under thecontrol of non-native promoters. Where it is desired that a pathwayoperate at a particular point in a cell cycle or during a fermentationrun, regulated or inducible promoters may used to replace the nativepromoter of the target gene(s). Similarly, in some cases the native orendogenous promoter may be modified to increase gene/gene clusterexpression. For example, endogenous promoters can be altered in vivo bymutation, deletion, and/or substitution (U.S. Pat. No. 5,565,350;Zarling et al., PCT/US93/03868).

Alternatively, where the sequences of the genes to be disrupted areknown, one of the most effective methods for gene down-regulation istargeted gene disruption, where foreign DNA is inserted into astructural gene so as to disrupt transcription. This can be affected bythe creation of genetic cassettes comprising the DNA to be inserted(often a genetic marker) flanked by sequences having a high degree ofhomology to a portion of the gene to be disrupted. Introduction of thecassette into the host cell results in insertion of the foreign DNA intothe structural gene via the native DNA replication mechanisms of thecell. (See for example Hamilton et al., J. Bacteriol., 171:46174622(1989); Balbas et al., Gene, 136:211-213 (1993); Gueldener et al.,Nucleic Acids Res., 24:2519-2524 (1996); and Smith et al., Methods Mol.Cell. Biol., 5:270-277(1996)).

Antisense technology is another method of down-regulating genes wherethe sequence of the target gene is known. To accomplish this, a nucleicacid segment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed.This construct is then introduced into the host cell and the antisensestrand of RNA is produced. Antisense RNA inhibits gene expression bypreventing the accumulation of mRNA encoding the protein of interest.The person skilled in the art will know that special considerations areassociated with the use of antisense technologies in order to reduceexpression of particular genes. For example, the proper level ofexpression of antisense genes may require the use of different chimericgenes utilizing different regulatory elements known to the skilledartisan.

Although targeted gene disruption and antisense technology offereffective means of down-regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence-based. For example, cells may be exposed to UV radiation andthen screened for the desired phenotype. Mutagenesis with chemicalagents is also effective for generating mutants and commonly usedsubstances include chemicals that affect nonreplicating DNA (e.g., HNO₂and NH₂OH), as well as agents that affect replicating DNA (e.g.,acridine dyes, notable for causing frameshift mutations). Specificmethods for creating mutants using radiation or chemical agents are welldocumented in the art. See, for example: Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed., (1989)Sinauer Associates: Sunderland, M A; or Deshpande, Mukund V., Appl.Biochem. Biotechnol. 36: 227-234 (1992).

Another non-specific method of gene disruption is the use oftransposable elements or transposons. Transposons are genetic elementsthat insert randomly in DNA but can be later retrieved on the basis ofsequence to determine where the insertion has occurred. Both in vivo andin vitro transposition methods are known. Both methods involve the useof a transposable element in combination with a transposase enzyme. Whenthe transposable element or transposon is contacted with a nucleic acidfragment in the presence of the transposase, the transposable elementwill randomly insert into the nucleic acid fragment. The technique isuseful for random mutagenesis and for gene isolation, since thedisrupted gene may be identified on the basis of the sequence of thetransposable element. Kits for in vitro transposition are commerciallyavailable (see, for example: The Primer Island Transposition Kit,available from Perkin Elmer Applied Biosystems, Branchburg, N.J., basedupon the yeast Ty1 element; The Genome Priming System, available fromNew England Biolabs, Beverly, Mass., based upon the bacterial transposonTn7; and the EZ::TN Transposon Insertion Systems, available fromEpicentre Technologies, Madison, Wis., based upon the Tn5 bacterialtransposable element).

Within the context of the present invention, it may be useful tomodulate the expression of the carotenoid biosynthetic pathway by anyone of the methods described above. For example, a number of genesencoding enzymes in the carotenoid pathway (crtE, crtX, crtY, crtl,crtB, crtZ, crtN, crtM, crtN1, crtN2, aid, sqs, etc.) are known, leadingto the production of the desired carotenoid. Thus, it may also be usefulto up-regulate the initial condensation of 3-carbon compounds (pyruvateand D-glyceraldehyde 3-phosphate) to increase the yield of the 5-carboncompound D-1-deoxyxylulose-5-phosphate (mediated by the dxs gene). Thiswould increase the flux of carbon entering the carotenoid biosyntheticpathway and permit increased production of astaxanthin. Alternatively(or in addition to), it may be desirable to knockout one ore more of thecrtN1, ald, or crtN2 genes leading to the synthesis of C₃₀ carotenoids,if the microbial host is capable of synthesizing these types ofcompounds. For example, an optimized Methylomonas sp. 16a strain hasbeen created containing a knockout of the native C₃₀ pathway, creating anon-pigmented strain suitable for engineering C₄₀ carotenoid prodution(U.S. Ser. No. 60/527,083; hereby incorporated by reference).

Methods of manipulating genetic pathways are common and well known inthe art. Selected genes in a particularly pathway may be upregulated ordown regulated by variety of methods. Additionally, competing pathwaysorganism may be eliminated or sublimated by gene disruption and similartechniques.

Industrial Production of Astaxanthin Using Recombinant Microorganisms

Where commercial production of astaxanthin is desired using the presentcrtOZ genes, a variety of culture methodologies may be applied. Forexample, large-scale production of the desired products (i.e.carotenoids and/or carotenoids biosynthesis enzymes) may be produced byboth batch and continuous culture methodologies.

A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to artificial alterations during the culturing process. Thus, atthe beginning of the culturing process the media is inoculated with thedesired organism or organisms and growth or metabolic activity ispermitted to occur adding nothing to the system. Typically, however, a“batch” culture is batch with respect to the addition of carbon sourceand attempts are often made at controlling factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

A variation on the standard batch system is the fed-batch system.Fed-batch culture processes are also suitable in the present inventionand comprise a typical batch system with the exception that thesubstrate is added in increments as the culture progresses. Fed-batchsystems are useful when catabolite repression is apt to inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the media. Measurement of the actual substrateconcentration in fed-batch systems is difficult and is thereforeestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases such as CO₂.Batch and fed-batch culturing methods are common and well known in theart and examples may be found in Brock (supra) or Deshpande (supra).

Commercial production of astaxanthin may also be accomplished with acontinuous culture. Continuous cultures are an open system where adefined culture media is added continuously to a bioreactor and an equalamount of conditioned media is removed simultaneously for processing.Continuous cultures generally maintain the cells at a constant highliquid phase density where cells are primarily in log phase growth.Alternatively, continuous culture may be practiced with immobilizedcells where carbon and nutrients are continuously added, and valuableproducts, by-products or waste products are continuously removed fromthe cell mass. Cell immobilization may be performed using a wide rangeof solid supports composed of natural and/or synthetic materials.

Continuous or semi-continuous culture allows for the modulation of onefactor or any number of factors that affect cell growth or end productconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen level at a fixed rate and allowall other parameters to moderate. In other systems a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomonosaccharides such as glucose and fructose, disaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally, the carbon substrate may also be one-carbonsubstrates such as methane or methanol, for which metabolic conversioninto key biochemical intermediates has been demonstrated (U.S. Ser. No.09/941,947; hereby incorporated by reference). In one embodiment, thecarbon substrate is selected from the group consisting of methane andmethanol and the recombinant host organisms is a methylotroph or amethanotroph. In addition to one and two carbon substratesmethylotrophic organisms are also known to utilize a number of othercarbon containing compounds such as methylamine, glucosamine and avariety of amino acids for metabolic activity. For example,methylotrophic yeast are known to utilize the carbon from methylamine toform trehalose or glycerol (Bellion et al., Microb. Growth C1-Compd.,[Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly,Don P. Publisher: Intercept, Andover, UK). Similarly, various species ofCandida will metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153:485-489 (1990)). Hence it is contemplated that the sourceof carbon utilized in the present invention may encompass a wide varietyof carbon containing substrates and will only be limited by the choiceof organism.

Methylotrophs and Methylomonas sp. 16a as Microbial Hosts

Although a number of carotenoids have been produced from recombinantmicrobial sources [e.g., E. coli and Candida utilis for production oflycopene (Farmer, W. R. and Liao, J. C., Biotechnol. Prog., 17: 57-61(2001); Wang et al., Biotechnol Prog., 16: 922-926 (2000); Misawa, N.and Shimada, H., J. Biotechnol., 59: 169-181 (1998); Shimada et al.,Appl. Environm. Microbiol., 64:2676-2680 (1998)]; E. coli, Candidautilis and Phaffia rhodozyma for production of β-carotene (Albrecht etal., Biotechnol. Lett., 21: 791-795 (1999); Miura et al., Appl.Environm. Microbiol., 64:1226-1229 (1998); U.S. Pat. No. 5,691,190); E.coli and Candida utilis for production of zeaxanthin (Albrecht et al.,supra; Miura et al., supra; E. coli and Phaffia rhodozyma for productionof astaxanthin (U.S. Pat. No. 5,466,599; U.S. Pat. No. 6,015,684; U.S.Pat. No. 5,182,208; U.S. Pat. No. 5,972,642); see also: U.S. Pat. No.5,656,472, U.S. Pat. No. 5,545,816, U.S. Pat. No. 5,530,189, U.S. Pat.No. 5,530,188, U.S. Pat. No. 5,429,939, and U.S. Pat. No. 6,124,113),these methods of producing carotenoids using various combinations ofdifferent crt genes suffer from low yields and reliance on relativelyexpensive feedstocks. Thus, it would be desirable to identify a methodthat produces high yields of carotenoids in a microbial host from aninexpensive feedstock.

There are a number of microorganisms that utilize single carbonsubstrates as their sole energy source. Such microorganisms are referredto herein as “C1 metabolizers”. These organisms are characterized by theability to use carbon substrates lacking carbon to carbon bonds as asole source of energy and biomass. These carbon substrates include, butare not limited to: methane, methanol, formate, formaldehyde, formicacid, methylated amines (e.g., mono-, di- and tri-methyl amine),methylated thiols, carbon dioxide, and various other reduced carboncompounds which lack any carbon-carbon bonds. In a particularembodiment, the carbon substrate is methanol and/or methane.

All C1 metabolizing microorganisms are generally classified asmethylotrophs. Methylotrophs may be defined as any organism capable ofoxidizing organic compounds that do not contain carbon-carbon bonds.However, facultative methylotrophs, obligate methylotrophs, and obligatemethanotrophs are all various subsets of methylotrophs. Specifically:

-   -   Facultative methylotrophs have the ability to oxidize organic        compounds which do not contain carbon-carbon bonds, but may also        use other carbon substrates such as sugars and complex        carbohydrates for energy and biomass. Facultative methylotrophic        bacteria are found in many environments, but are isolated most        commonly from soil, landfill and waste treatment sites. Many        facultative methylotrophs are members of the β and γ subgroups        of the Proteobacteria (Hanson et al., Microb. Growth C1        Compounds., [Int. Symp.], 7^(th) (1993), pp 285-302. Murrell, J.        Collin and Don P. Kelly, eds. Intercept: Andover, UK; Madigan et        al., Brock Biology of Microorganisms, 8^(th) ed., Prentice Hall:        Upper Saddle River, N.J. (1997)).    -   Obligate methylotrophs are those organisms that are limited to        the use of organic compounds that do not contain carbon-carbon        bonds for the generation of energy.    -   Obligate methanotrophs are those obligate methylotrophs that        have the distinct ability to oxidize methane.

Additionally, the ability to utilize single carbon substrates is notlimited to bacteria but extends also to yeasts and fungi. A number ofyeast genera are able to use single carbon substrates as energy sourcesin addition to more complex materials (i.e., the methylotrophic yeasts).

Although a large number of these methylotrophic organisms are known, fewof these microbes have been successfully harnessed in industrialprocesses for the synthesis of materials. And, although single carbonsubstrates are cost-effective energy sources, difficulty in geneticmanipulation of these microorganisms as well as a dearth of informationabout their genetic machinery has limited their use primarily to thesynthesis of native products.

Despite these hardships, many methanotrophs contain an inherentisoprenoid pathway which enables these organisms to synthesize pigmentsand provides the potential for one to envision engineering thesemicroorganisms for production of other non-endogenous isoprenoidcompounds. Since methanotrophs can use single carbon substrates (i.e.,methane and/or methanol) as an energy source, it could be possible toproduce carotenoids at low cost in these organisms. Examples wherein amethanotroph was engineered for production of β-carotene are describedin U.S. Ser. No. 09/941,947 and U.S. Ser. No. 60/527,083.

In the present invention, methods are provided for the expression ofgenes involved in the biosynthesis of astaxanthin in microorganisms thatare able to use single carbon substrates as a sole energy source. Thehost microorganism may be any C1 metabolizer that has the ability tosynthesize β-carotene as a metabolic precursor for astaxanthin. Morespecifically, facultative methylotrophic bacteria suitable in thepresent invention include, but are not limited to: Methylophilus,Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter,Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, andPseudomonas. Specific methylotrophic yeasts useful in the presentinvention include, but are not limited to: Candida, Hansenula, Pichia,Torulopsis, and Rhodotorula. And, exemplary methanotrophs are includedin, but are not limited to, the genera Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocyctis, Methylomicrobium, andMethanomonas.

Of particular interest in the present invention are high growth obligatemethanotrophs having an energetically favorable carbon flux pathway. Forexample, a specific strain of methanotroph has been discovered havingseveral pathway features that makes it particularly useful for carbonflux manipulation. This strain is known as Methylomonas sp. 16a (ATCCPTA 2402) (U.S. Pat. No. 6,689,601; hereby incorporated by reference);and, this particular strain and other related methylotrophs arepreferred microbial hosts for expression of the gene products of thisinvention, useful for the production of C₄₀ carotenoids (U.S. Ser. No.09/941,947).

Methylomonas sp. 16a naturally produces C₃₀ carotenoids. Odom et al.have reported that expression of C₄₀ carotenoid genes in Methylomonas16a produced a mixture of C₃₀ and C₄₀ carotenoids (U.S. Ser. No.09/941,947). Several of the genes involved in C₃₀ carotenoid productionin this strain have been identified including (but not limited to) thecrtN1, ald, crtN2, and crtN3 genes. Disruption of the crtN1/ald genes orthe promoter driving expression of the crtN1/ald/crtN2 gene clustercreated various non-pigmented mutants (“white mutants”) more suitablefor C₄₀ carotenoid production (U.S. Ser. No. 60/527,083, herebyincorporated by reference). For example, a non-pigmented Methylomonassp. 16a strain “MWM1000” was created by disrupting the ald and crtN1genes (U.S. Ser. No. 60/527,083).

Transformation of C1 Metabolizing Bacteria

Techniques for the transformation of C1 metabolizing bacteria are notwell developed, although general methodology that is utilized for otherbacteria, which is well known to those of skill in the art, may beapplied. Electroporation has been used successfully for thetransformation of: Methylobacterium extorquens AM1 (Toyama, H., et al.,FEMS Microbiol. Lett., 166:1-7 (1998)), Methylophilus methylotrophus AS1(Kim, C. S., and Wood, T. K., Appl. Microbiol. Biotechnol., 48: 105-108(1997)), and Methylobacillus sp. strain 12S (Yoshida, T., et al.,Biotechnol. Lett., 23: 787-791 (2001)). Extrapolation of specificelectroporation parameters from one specific C1 metabolizing utilizingorganism to another may be difficult, however, as is well to known tothose of skill in the art.

Bacterial conjugation, relying on the direct contact of donor andrecipient cells, is frequently more readily amenable for the transfer ofgenes into C1 metabolizing bacteria. Simplistically, this bacterialconjugation process involves mixing together “donor” and “recipient”cells in close contact with one another. Conjugation occurs by formationof cytoplasmic connections between donor and recipient bacteria, withdirect transfer of newly synthesized donor DNA into the recipient cells.As is well known in the art, the recipient in a conjugation is definedas any cell that can accept DNA through horizontal transfer from a donorbacterium. The donor in conjugative transfer is a bacterium thatcontains a conjugative plasmid, conjugative transposon, or mobilizableplasmid. The physical transfer of the donor plasmid can occur in one oftwo fashions, as described below:

-   -   In some cases, only a donor and recipient are required for        conjugation. This occurs when the plasmid to be transferred is a        self-transmissible plasmid that is both conjugative and        mobilizable (i.e., carrying both tra genes and genes encoding        the Mob proteins). In general, the process involves the        following steps: 1.) Double-strand plasmid DNA is nicked at a        specific site in on T; 2.) A single-strand DNA is released to        the recipient through a pore or pilus structure; 3.) A DNA        relaxase enzyme cleaves the double-strand DNA at on T and binds        to a release 5′ end (forming a relaxosome as the intermediate        structure); and 4.) Subsequently, a complex of auxiliary        proteins assemble at on T to facilitate the process of DNA        transfer.    -   Alternatively, a “triparental” conjugation is required for        transfer of the donor plasmid to the recipient. In this type of        conjugation, donor cells, recipient cells, and a “helper”        plasmid participate. The donor cells carry a mobilizable plasmid        or conjugative transposon. Mobilizable vectors contain an on T,        a gene encoding a nickase, and have genes encoding the Mob        proteins; however, the Mob proteins alone are not sufficient to        achieve the transfer of the genome. Thus, mobilizable plasmids        are not able to promote their own transfer unless an appropriate        conjugation system is provided by a helper plasmid (located        within the donor or within a “helper” cell). The conjugative        plasmid is needed for the formation of the mating pair and DNA        transfer, since the plasmid encodes proteins for transfer (Tra)        that are involved in the formation of the pore or pilus.

Examples of successful conjugations involving C1 metabolizing bacteriainclude the work of: Stolyar et al. (Mikrobiologiya 64(5): 686-691(1995)); Motoyama et al. (Appl. Micro. Biotech. 42(1): 67-72 (1994));Lloyd et al. (Archives of Microbiology 171(6): 364-370 (1999)); U.S.Ser. No. 09/941,947; U.S. Ser. No. 60/527,083; and U.S. Ser. No.60/527,877, hereby incorporated by reference.

Recombinant Expression of crtOZ—Plants

Plants and algae are also known to produce carotenoid compounds, such asastaxanthin. The nucleic acid fragments of the instant invention may beused to create transgenic plants having the ability to express themicrobial protein. Preferred plant hosts will be any variety that willsupport a high production level of the instant proteins. Suitable greenplants will include but are not limited to soybean, rapeseed (Brassicanapus, B. campestris), pepper, sunflower (Helianthus annus), cotton(Gossypium hirsutum), corn, tobacco (Nicotiana tabacum), alfalfa(Medicago sativa), wheat (Triticum sp), barley (Hordeum vulgare), oats(Avena sativa, L), sorghum (Sorghum bicolor), rice (Oryza sativa),Arabidopsis, cruciferous vegetables (broccoli, cauliflower, cabbage,parsnips, etc.), melons, carrots, celery, parsley, tomatoes, potatoes,strawberries, peanuts, grapes, grass seed crops, sugar beets, sugarcane, beans, peas, rye, flax, hardwood trees, softwood trees, and foragegrasses. Algal species include but not limited to commerciallysignificant hosts such as Spirulina, Haemotacoccus, and Dunalliela.Production of the carotenoid compounds may be accomplished by firstconstructing chimeric genes of present invention in which the codingregion are operably linked to promoters capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric genes may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mustalso be provided. The instant chimeric genes may also comprise one ormore introns in order to facilitate gene expression.

Any combination of any promoter and any terminator capable of inducingexpression of a coding region may be used in the chimeric geneticsequence(s). Some suitable examples of promoters and terminators includethose from nopaline synthase (nos), octopine synthase (ocs) andcauliflower mosaic virus (CaMV) genes. One type of efficient plantpromoter that may be used is a high level plant promoter. Suchpromoters, in operable linkage with the genetic sequences or the presentinvention should be capable of promoting expression of the present geneproduct. High level plant promoters that may be used in this inventioninclude the promoter of the small subunit (ss) of theribulose-1,5-bisphosphate carboxylase from example from soybean(Berry-Lowe et al., J. Molecular and App. Gen., 1:483-498 1982)), andthe promoter of the chlorophyll a/b binding protein. These two promotersare known to be light-induced in plant cells (see, for example, GeneticEngineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum,N.Y. (1983), pages 29-38; Coruzzi, G. et al., The Journal of BiologicalChemistry, 258:1399 (1983), and Dunsmuir, P. et al., Journal ofMolecular and Applied Genetics, 2:285 (1983)).

Plasmid vectors comprising the instant chimeric genes can thenconstructed. The choice of plasmid vector depends upon the method thatwill be used to transform host plants. The skilled artisan is well awareof the genetic elements that must be present on the plasmid vector inorder to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA blots (Southern, J. Mol.Biol. 98, 503, (1975)). Northern analysis of mRNA expression (Kroczek,J. Chromatogr. Biomed. Appl., 618 (1-2) (1993) 133-145), Westernanalysis of protein expression, or phenotypic analysis.

For some applications it will be useful to direct the instant proteinsto different cellular compartments. It is thus envisioned that thechimeric genes described above may be further supplemented by alteringthe coding sequences to encode enzymes with appropriate intracellulartargeting sequences such as transit sequences (Keegstra, K., Cell,56:247-253 (1989)), signal sequences or sequences encoding endoplasmicreticulum localization (Chrispeels, J. J., Ann. Rev. Plant Phys. PlantMol. Biol., 42:21-53 (1991)), or nuclear localization signals (Raikhel,N., Plant Phys., 100:1627-1632 (1992)) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future that areuseful in the invention.

In Vitro Bio-Conversion of Carotenoids

Alternatively, it is possible to carry out the bioconversions of thepresent application in vitro. Where substrates for the present CrtOketolase and CrtZ hydroxylase are not synthesized by the host cell, itwill be possible to add the substrate(s) exogenously. In this embodimentthe suitable carotenoid substrate may be solubilized with mild detergent(e.g., DMSO) or mixed with phospholipid vesicles. To assist in transportinto the cell, the host cell may optionally be permeabilized with asuitable solvent such as toluene. Methods for this type of in-vitrobio-conversion of carotenoid substrates has basis in the art (see forexample: Hundle, B. S., et al., FEBS, 315:329-334 (1993); and Bramley,P. M., et al., Phytochemistry, 26:1935-1939 (1987)).

Protein Engineering CrtO Ketolases and CrtZ Hydroxylases

The present nucleic acid fragments encoding the CrtO ketolases and CrtZhydroxylases were protein engineered by subjecting the instand genes tomutational conditions using error-prone PCR ((Melnikov et al., NucleicAcids Research, 27(4):1056-1062 (1999); Leung et al., Techniques,1:11-15 (1989); and Zhou et al., Nucleic Acids Res., 19:6052-6052(1991)). It is contemplated that the present crtOZ genes may be furtherengineered to produce gene products having further enhanced or alteredactivity. Alternate methods of mutating genes and selecting for mutantsare known including, but not limited to: 1.) site-directed mutagenesis(Coombs et al., Proteins (1998), pp 259-311, 1 plate. Angeletti, RuthHogue, Ed., Academic: San Diego, Calif.); and 2.) “gene-shuffling” (U.S.Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721;and U.S. Pat. No. 5,837,458 or any similar means of promotingrecombinogenic activity between nucleic acids (see for example Tang etal., U.S. Ser. No. 10/374,366; hereby incorporated by reference)).

The method of gene shuffling has the advantage of facile implementation,high rate of mutagenesis, and ease of screening. The process of geneshuffling involves the restriction endonuclease cleavage of a gene ofinterest into fragments of specific size in the presence of additionalpopulations of DNA fragments having regions of similarity or differenceto the gene of interest. This pool of fragments will then be denaturedand reannealed to create a mutated gene. The mutated gene is thenscreened for altered activity.

The present sequences may be mutated and screened for altered orenhanced activity by this method. The sequences should bedouble-stranded and can be of various lengths ranging from 50 bp to 10kB. The sequences may be randomly digested into fragments ranging fromabout 10 bp to 1000 bp, using restriction endonucleases well known inthe art (Maniatis, supra). In addition to the instant microbialsequences, populations of fragments that are hybridizable to all orportions of the microbial sequence may be added. Similarly, a populationof fragments that are not hybridizable to the instant sequences may alsobe added. Typically these additional fragment populations are added inabout a 10 to 20-fold excess by weight as compared to the total nucleicacid. Generally, if this process is followed, the number of differentspecific nucleic acid fragments in the mixture will be about 100 toabout 1000. The mixed population of random nucleic acid fragments aredenatured to form single-stranded nucleic acid fragments and thenreannealed. Only those single-stranded nucleic acid fragments havingregions of homology with other single-stranded nucleic acid fragmentswill reanneal. The random nucleic acid fragments may be denatured byheating. One skilled in the art could determine the conditions necessaryto completely denature the double-stranded nucleic acid. Preferably, thetemperature is from about 80° C. to 100° C. The nucleic acid fragmentsmay be reannealed by cooling. Preferably the temperature is from about20° C. to 75° C. Renaturation can be accelerated by the addition ofpolyethylene glycol (“PEG”) or salt. A suitable salt concentration mayrange from 0 mM to 200 mM. The annealed nucleic acid fragments are thenincubated in the presence of a nucleic acid polymerase and dNTPs (i.e.,dATP, dCTP, dGTP and dTTP). The nucleic acid polymerase may be theKlenow fragment, the Taq polymerase or any other DNA polymerase known inthe art. The polymerase may be added to the random nucleic acidfragments prior to annealing, simultaneously with annealing or afterannealing. The cycle of denaturation, renaturation and incubation in thepresence of polymerase is repeated for a desired number of times.Preferably, the cycle is repeated from about 2 to 50 times, morepreferably the sequence is repeated from 10 to 40 times. The resultingnucleic acid is a larger double-stranded polynucleotide ranging fromabout 50 bp to about 100 kB and may be screened for expression andaltered activity by standard cloning and expression protocols (Maniatis,supra).

Furthermore, a hybrid protein can be assembled by fusion of functionaldomains using the gene shuffling (exon shuffling) method (Nixon et al.,Proc. Natl. Acad. Sci., 94:1069-1073 (1997)). The functional domain ofthe instant gene(s) can be combined with the functional domain of othergenes to create novel enzymes with desired catalytic function.

In addition to the methods exemplified above (which are designed todirectly mutagenize the crtOZ gene clusters encoding CrtO ketolases andCrtZ hydroxylases), traditional methods of creating mutants could beutilized for the purposes described herein. For example, wild-type cellshaving carotenoid ketolase and carotenoid hydroxylase activity may beexposed to a variety of agents such as radiation or chemical mutagensand then screened for the desired phenotype. When creating mutationsthrough radiation either ultraviolet (UV) or ionizing radiation may beused. Suitable short wave UV wavelengths for genetic mutations will fallwithin the range of 200 nm to 300 nm, where 254 nm is preferred. UVradiation in this wavelength principally causes changes within nucleicacid sequence from guanidine and cytosine to adenine and thymidine.Since all cells have DNA repair mechanisms that would repair most UVinduced mutations, agents such as caffeine and other inhibitors may beadded to interrupt the repair process and maximize the number ofeffective mutations. Long wave UV mutations using light in the 300 nm to400 nm range are also possible; but this range is generally not aseffective as the short wave UV light, unless used in conjunction withvarious activators (such as psoralen dyes) that interact with the DNA.Likewise, mutagenesis with chemical agents is also effective forgenerating mutants and commonly used substances include chemicals thataffect nonreplicating DNA (such as HNO₂ and NH₂OH), as well as agentsthat affect replicating DNA (such as acridine dyes, notable for causingframeshift mutations). Specific methods for creating mutants usingradiation or chemical agents are well documented in the art. See, forexample, Brock (supra) or Deshpande (supra).

Method of gene mutation preferred herein involve Error Prone PCR.Accordingly, the present crtOZ genes were simultaneously mutated (i.e.exposed to mutational conditions) using error-prone PCR to create geneclusters encoding CrtO/CrtZ enzymes having the ability to produceastaxanthin. In one embodiment, a method to produce match carotenoidketolase and carotenoid hydroxylase enzymes exhibiting improvedproduction of astaxanthin is provided. The method is comprised ofsimultaneously mutating (or at least exposing the starting genes tomutational conditions) any carotenoid hydroxylase (CrtZ or CrtR) andcarotenoid ketolase (CrtO, CrtW/bkt) using any well known proteinengineering techniques (error-prone PCR, gene shuffling, randommutagenesis, etc.) to produce hydroxylase/ketolase combinations havingan improved ability to produce astaxanthin. The resultingketolase/hydroxylase gene clusters are then simultaneously transformedand expressed in recombinant host cells capable of producing β-carotene.Transformants exhibiting improved astaxanthin production (assessedeither by visually screening or any common analytical method such asHPLC) are then evaluated for mutations accounting for the structuralchanges responsible for improved astaxanthin production. The matchedcarotenoid ketolase/carotenoid hydroxylase genes exhibiting improvementsin astaxanthin production can be selected as the “starting genes” foradditional rounds of protein engineering. In an optional embodiment, thecrtO and crtZ genes can be individually mutated and then coexpressed ina recombinant host cell capable of producing β-carotene in order toevaluate the combination's ability to produce astaxanthin. However,simultaneous mutantion and coexpression of the mutated genes ispreferable, thereby being able to select (in a single step) crtOZ geneclusters having optimal astaxanthin synthesizing activity.

Irrespective of the method of mutagenesis, the crtOZ genes may beevolved such that the enzymes have an increase in astaxanthin synthesisactivity. The increase in astaxanthin synthesis activity can be measuredusing a variety of techniques known in the art. In the presentinvention, a simple measurement of astaxanthin production in thepresence of excess substrate (i.e. β-carotene) under identical reactionconditions will typically be suitiable to identify enzymes capable ofproviding a higher percentage yield of a astaxanthin.

EXAMPLES

General Methods:

Procedures required for PCR amplification, DNA modifications by endo-and exonucleases for generating desired ends for cloning of DNA,ligation, and bacterial transformation are well known in the art.Standard molecular cloning techniques used here are well known in theart and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T.Molecular Cloning: A Laboratory Manual, 2^(nd) ed.; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter “Maniatis”); andby Silhavy, T. J., Bennan, M. L. and Enquist, L. W. Experiments withGene Fusions; Cold Spring Harbor Laboratory: Cold Spring, N.Y., 1984 andby Ausubel et al., Current Protocols in Molecular Biology; GreenePublishing and Wiley-Interscience; 1987.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology; Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds., American Society for Microbiology: Washington,D.C., 1994 or by Brock, T. D.; Biotechnology: A Textbook of IndustrialMicrobiology, 2nd ed.; Sinauer Associates: Sunderland, M A, 1989. Allreagents, restriction enzymes and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

PCR reactions were run on GeneAMP PCR System 9700 using Amplitaq orAmplitaq Gold enzymes (PE Applied Biosystems, Foster City, Calif.),unless otherwise specified. The cycling conditions and reactions werestandardized according to the manufactures' instructions.

The meaning of abbreviations is as follows: “min”means minute(s), “h”means hour(s), “μL” means microliter, “mL” means milliliters, “L” meansliters, “cm” means centimeters, “nm” means nanometers, “mM” meansmillimolar, “kB” means kilobases, and “kV” means kilovolts.

Example 1 Selection of the crtOZ Plasmid for Protein EngineeringConstruction of two plasmids containing different crtOZ genes

The crtO gene isolated from Rhodococcus erythropolis AN12 has previouslybeen engineered (U.S. Ser. No. 60/577,970) to increase the ketolaseactivity. One of the mutants, crtO-SHU001, produced over 90%canthaxanthin in E. coli when coexpressed with {overscore (β)}-carotenesynthesis genes. This crtO was chosen to pair with crtZ for astaxanthinproduction. The crtO-SHU001 gene (SEQ ID NOs: 1 and 3) was PCR-amplifiedfrom pDCQ320-SHU001 plasmid DNA (U.S. Ser. No. 60/577,970), usingforward primer crtO-SHU001-F 5′-ACTAGTAAGGAGGAATAAACCATGAGCGCA-3′ (SEQID NO: 3) and reverse primer crtO-SHU001-R5′-TGTACAGCTAGCTCACGAGCGGCTCGAACGACGCAT-3′ (SEQ ID NO: 4). Underlinedare restriction sites for Spe I, Nhe I and BrsG I. The ˜1.6 kb PCRproduct was gel purified and cloned into pTrcHis2-Topo vector, resultingin plasmid pDCQ353. The ˜1.6 kb SpeI I/BrsG I fragment of pDCQ353 DNAcontaining the crtO-SHU001 gene was cloned to the pDCQ334 plasmid(pDCQ334 is a plasmid comprised of a crtWZEidiYIB gene cluster preparedby cloning the codon optimized (for Methylomonas 16a) Agrobacteriumaurantiacum crtWZ genes upstream of the Pantoea agglomerans DC404β-carotene gene cluster; see U.S. Ser. No. 60/527,083 and U.S. Ser. No.10/808,807; herby incorporated by reference) digested with SpeI/BsrGI toremove crtWZ, which resulted in pDCQ354 containing crtOEidiYIB. E. colicells containing pDCQ354 were orange and produced almost exclusivelycanthaxanthin.

The crtZ genes from two different sources were compared to see how wellthey function with the CrtO (encoded by crtO-SHU001) for astaxanthinproduction. The crtZ from DC263 (U.S. Ser. No. 60/601,947; SEQ ID NOs: 5and 6) was amplified using primers crtZ-263_F2:5′-TCTAGAAAGGAGGAATAAACCATGTCCTGGCCGACGATGATC-3′ (SEQ ID NO: 7) andcrtZ-263_R2: 5′-ACTAGTCAGGCGCCGTTGCTGGATGA-3′ (SEQ ID NO: 8). The 507 bpPCR product was cloned into pTrcHis2-TOPO vector resulting pDCQ352. ThecrtZ from DC260 (U.S. Ser. No. 10/808,979; SEQ ID NOs: 9 and 10) wasamplified using forward primer crtZ-DC260-F5′-ACTAGTAAGGAGGAATAAACCATGCTCTGGTTATGGAACGTGC-3′ (SEQ ID NO: 11) andreverse primer crtZ-DC260-R 5′-ACTAGTTCACTTCGCGTGTGTCTCGTC-3′ (SEQ IDNO: 12). The 561 bp PCR product was cloned into pTrcHis2-Topo vector,resulting in plasmid pDCQ355. Underlined are restriction sites for Spe Iand XbaI. The XbaI-SpeI fragment containing DC263 crtZ from pDCQ352 andthe SpeI fragment containing DC260 crtZ from pDCQ355 were cloned intothe SpeI site of pDCQ354, resulting pDCQ356 and pDCQ357, respectively.

Carotenoid Analysis of Cells Containing pDCQ356 or PDCQ357

HPLC analysis was performed on cells containing these plasmids. Cellswere pelleted by centrifugation at 4000 g for 15 min, and the cellpellets were extracted with 1-2 mL acetone. The extraction was driedunder nitrogen and redisolved in 0.5 mL of 50% acetone +50% methanol.The extraction was filtered with an Acrodisc® CR25 mm syringe filter(Pall Corporation, Ann Arbor, Mich.) for HPLC analysis using an AgilentSeries 1100 LC/MSD SI (Agilent, Foster City, Calif.).

Samples (20 μL) were loaded onto a 150 mm×4.6 mm ZORBAX C18 (3.5 μmparticles) column (Agilent Technologies, Inc.). The column temperaturewas kept at 40° C. The flow rate was 1 mL/min, while the solvent runningprogram used was 0-2 min: 95% Buffer A and 5% Buffer B;

2-10 min: linear gradient from 95% Buffer A and 5% Buffer B to 60%Buffer A and 40% Buffer B;

10-12 min: linear gradient from 60% Buffer A and 40% Buffer B to 50%Buffer A and 50% Buffer B;

12-18 min: 50% Buffer A and 50% Buffer B; and,

18-20 min: 95% Buffer A and 5% Buffer B.

Buffer A was 95% acetonitrile and 5% dH₂O; Buffer B was 100%tetrahydrofuran (THF). The mass spectrometer was scanned from 250 to 900e/z in APCI (Atomospheric Pressure Chemical Ionization) mode with thefragmentation voltage at 70 V. No astaxanthin was produced in E. colicontaining either of the plasmids. These E. coli cells were yellow andproduced predominantly zeaxanthin and trace amounts of ketocarotenoids.These two plasmids were transferred into Methylomonas sp. 16a(non-pigmented MWM1200 strain; U.S. Ser. No. 60/527,083; herebyincorporated by reference) by tri-parental conjugation. Astaxanthin wasnot produced in Methylomonas tranformants containing either plasmid.Methylomonas containing pDCQ356 produced carotenoids containing 69%zeaxanthin and 26% adonixanthin. Methylomonas containing pDCQ357produced carotenoids containing 80% zeaxanthin and 11% adonixanthin.Since higher amount of adonixanthin intermediate was produced bypDCQ356, this plasmid pDCQ356 was chosen for protein engineering toproduce astaxanthin.

Example 2 Making Mutant Libraries

Error-Prone PCR:

The plasmid pDCQ356 was used as a template for error-prone PCR. Theinsert containing the crtOZ genes (SEQ ID NO: 1 and SEQ ID NO: 5;respectively) can be removed from the construct using BsrG I and Spe Idigestion. A random mutant library targeting the crtOZ genes was madeusing error-prone PCR. The following primers were used to amplify theinserts by error-prone PCR: (SEQ ID NO: 13) 334F1 5′-GCA GCG TGC AGC TCATGC AGT TC-3′ (SEQ ID NO: 14) 334r1 5′-CCA GAC CGT TCA GCT GGA TATTAC-3′

A Clontech mutagenesis kit (Clontech Laboratories, Inc., Palo Alto,Calif.) was used for performing error-prone PCR. The following conditionwas used for preparing error-prone PCR reaction mixture: TABLE 1Conditions for Error-prone PCR using Clontech Mutagenesis Kit Volumes(μL) PCR grade water 37 10x AdvanTaq Plus Buff. 5 MnSO₄ (8 mM) 3 dGTP (2mM) 1 50x Diversify dNTP Mix 1 Primer mix 0 Template DNA 1 AdvanTaq PlusPolym. 1

The thermal cycling reaction was carried out according to themanufacturer's instructions. The PCR products were digested with BsrGI/Spe I.

Mutant Library Construction:

To prepare the vector, the template plasmid (pDCQ356) was digested withBsrG I and Spe I to remove the insert. The digested vector was purifiedfrom the agarose gel. The BsrG I/Spe I-digested error-prone PCR productswere then ligated with the BsrG I/Spe 1-digested vectors. After ethanolprecipitation, the ligation mixture was ready for the transformation.

The ligation mixture was first transformed intoElectroporation-Competent E. coli 10G cells (Lucigen Corp., Middleton,Wis.) by electroporation. The cells were plated onto LB plates in thepresence of kanamycin and incubated overnight at 37° C. The mutantcolonies were ready for high-throughput screening.

DNA Sequence Analysis of the Mutant Libraries:

Ten mutant colonies from each library were randomly picked for DNAsequencing analyses. The mutant genes were sequenced on an ABI 377automated sequencer (Applied Biosystems, Foster City, Calif.), and thedata managed using Vector NTI program (InforMax, Inc., Bethesda, Md.).Most of the mutations were base substitutions, the frequency ofdeletions and insertions in the mutant libraries was very low. Varioustypes of base substitution were present in these mutants, indicatingthere was no bias for the mutation type. The mutation rate wasapproximately 1-5 point mutations per kB.

Example 3 Screening the Mutant Libraries and Identifying the Hits

The color of cells containing pDCQ356 was light yellow. The color of thecells producing astaxanthin is red-orange. The cells that make differentpercentages of astaxanthin show slightly different levels ofpigmentation. Therefore, the mutant colonies that produce differentamounts of astaxanthin can be distinguished visually. Approximately10,000-20,000 mutant colonies from the mutant library were screenedvisually. Nine putative hits were streaked on Agar plates.

A follow-up confirmation assay was performed by HPLC analysis. E. coli10G cells containing pDCQ356 and its mutant derivatives were grown in 25ml LB with 50 μg/mL kanamycin at 30° C.; shaking for two days. Cellswere harvested by centrifugation and extracted with 50% acetone and 50%methanol. HPLC analysis of the carotenoids was performed as described inExample 1. Two of the nine crtOZ mutants produced astaxanthin as shownin Table 2. TABLE 2 HPLC Confirmation Analysis Results pDCQ356Carotenoids (starting genes) pDCQ356M4003 pDCQ356M4005 astaxanthin 0%26% 20% adonixanthin 5% 4% 31% zeaxanthin 80% <1% 8% adonirubin 0% 21%12% canthaxanthin 0% 44% 18%The data in Table 2 showed that the percentage yield of carotenoids. Thestarting construct (pDCQ356) did not make any astaxanthin. However, twomutants made 20-26% of astaxanthin and other intermediates. The rest ofputative hits did not make any astaxanthin, but produced canthaxanthinand echinenone.

Example 4 DNA Sequence Analysis of the Mutant Genes

The mutant genes were sequenced on an ABI377 automated sequencer(Applied Biosystem, Foster City, Calif.), and the data managed usingVector NTI program (InforMax, Inc., Bethesda, Md.). Analysis of themutants, followed by comparison with the starting genes, indicated thatthe mutant genes contained the following point mutations: TABLE 3 DNAsequence analysis of mutant genes Strain Starting Gene(s)/Mutations356M4003 crtO-SHU0014: (SEQ ID NOs. 15-19) GCA(Ala16) to GCT(Ala)GGG(Gly203) to GGA(Gly) CTC(Leu305) to CTT(Leu) CrtZ DC263: CTG(Leu53)to CCG(Pro) ACG(Thr84) to ACT(Thr) ACA(Thr128) to ACC(Thr) 356M4005crtO-SHU004: (SEQ ID NOs. 20-24) GCA(Ala190) to GTA(Val) GTT(Val277) toGTC(Val) CTC(Leu305) to CTT(Leu) CrtZ DC263: TTC(Phe91) to TCC(Ser)GTG(Val140) to GGG(Gly)Except for the silent mutations, all the mutations were the amino acidsubstitutions.

Example 5 Performance of Mutant Genes in Methylomonas

Plasmid pDCQ356 and the mutant derivatives were transferred intoMethylomonas sp. 16a (MWM1200 strain; U.S. Ser. No. 60/527,083) bytri-parental conjugal mating. The E. coli helper strain containingpRK2013 (ATCC No. 37159) and the E. coli XL1 BlueMRF′ donor strainscontaining the plasmid were each grown overnight in LB medium containingkanamycin (50 μg/mL), washed three times in LB, and resuspended in avolume of LB representing approximately a 60-fold concentration of theoriginal culture volume.

The Methylomonas 16a recipient MWM1200 was grown using the generalconditions described in WO 02/18617. Briefly, this involves growingMethylomonas 16a in serum stoppered Wheaton bottles (Wheaton Scientific,Wheaton Ill.) using a gas/liquid ratio of at least 8:1 (i.e., 20 mL ofNitrate liquid “BTZ-3” media in 160 mL total volume) at 30° C. withconstant shaking.

Nitrate Medium for Methylomonas 16A

Nitrate liquid medium, also referred to herein as “defined medium” or“BTZ-3” medium is comprised of various salts mixed with Solution 1 asindicated below (Tables 4 and 5) or where specified the nitrate isreplaced with 15 mM ammonium chloride. Solution 1 provides thecomposition for 100-fold concentrated stock solution of trace minerals.TABLE 4 Solution 1* Conc. MW (mM) g per L Nitriloacetic acid 191.1 66.912.8 CuCl₂ × 2H₂O 170.48 0.15 0.0254 FeCl₂ × 4H₂O 198.81 1.5 0.3 MnCl₂ ×4H₂O 197.91 0.5 0.1 CoCl₂ × 6H₂O 237.9 1.31 0.312 ZnCl₂ 136.29 0.73 0.1H₃BO₃ 61.83 0.16 0.01 Na₂MoO₄ × 2H₂O 241.95 0.04 0.01 NiCl₂ × 6H₂O 237.70.77 0.184*Mix the gram amounts designated above in 900 mL of H₂O, adjust to pH =7, and add H₂O to an end volume of 1 L. Keep refrigerated.

TABLE 5 Nitrate liquid medium (BTZ-3)** Conc. MW (mM) g per L NaNO₃84.99 10 0.85 KH₂PO₄ 136.09 3.67 0.5 Na₂SO₄ 142.04 3.52 0.5 MgCl₂ × 6H₂O203.3 0.98 0.2 CaCl₂ × 2H₂O 147.02 0.68 0.1 1 M HEPES (pH 7) 238.3 50 mLSolution 1 10 mL**Dissolve in 900 mL H₂O. Adjust to pH = 7, and add H₂O to give 1 L.For agar plates: Add 15 g of agarose in 1 L of medium, autoclave, letcool down to 50° C., mix, and pour plates.

The standard gas phase for cultivation contains 25% methane in air.Using these conditions, the recipient was cultured for 48 h in BTZ-3medium, washed three times in BTZ-3, and resuspended in a volume ofBTZ-3 representing a 150-fold concentration of the original culturevolume.

The donor, helper, and recipient cell pastes were then combined inratios of 1:1:2, respectively, on the surface of BTZ-3 agar platescontaining 0.5% (w/v) yeast extract. Plates were maintained at 30° C. in25% methane for 16-72 h to allow conjugation to occur, after which thecell pastes were collected and resuspended in BTZ-3. Dilutions wereplated on BTZ-3 agar containing kanamycin (50 μg/mL) and incubated at30° C. in 25% methane for up to 1 week. Transconjugants were streakedonto BTZ-3 agar with kanamycin (50 μg/mL) for isolation.

For analysis of carotenoid composition, Methylomonas transconjugantswere cultured in a 24-well blocks (Qiagen catalog no. 19583) with eachwell containing 1 mL BTZ-3 containing kanamycin (50 μg/mL). The blockwas covered with Airpore™ film (Qiagen) and incubated in an AnaeroPack™System (Mitsubishi Gas Chemical Co., Inc., Japan) filled with 25%methane as the sole carbon source. The AnaeroPack™ was shaking at 250rpm for 2-3 days at 30° C. The cells were harvested by centrifugationand the pellets were extracted and carotenoid content was analyzed byHPLC, as described in Example 1. Table 6 summarized the results: TABLE 6HPLC analysis results pDCQ356 Carotenoids (starting genes) pDCQ356M4003pDCQ356M4005 astaxanthin 0% 5% 37% adonixanthin 26% <1% 5% zeaxanthin69% <1% <1% adonirubin 0% 11% 11% canthaxanthin 0% 70% 45%

1. An isolated nucleic acid molecule encoding at least one carotenoidketolase and at least one carotenoid hydroxylase, said nucleic acidmolecule comprising: a) a nucleic acid fragment encoding a carotenoidketolase having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 17 and SEQ ID NO. 22; and b) a isolated nucleicacid fragment encoding a carotenoid hydroxylase having an amino acidsequence selected from the group consisting of SEQ ID NO: 19 and SEQ IDNO: 24: or an isolated nucleic acid molecule completely complementary tothe nucleic acid molecule comprising the elements of (a) and (b).
 2. Anisolated nucleic acid molecule encoding at least one carotenoid ketolaseand at least one carotenoid hydroxylase, said nucleic acid moleculecomprising: a) a nucleic acid fragment encoding a carotenoid ketolasehaving a nucleic acid sequence selected from the group consisting of SEQID NO: 16 and SEQ ID NO: 21; and b) a nucleic acid fragment encoding acarotenoid hydroxylase having a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 18 and SEQ D NO: 23; or an isolatednucleic acid molecule completely complementary to the nucleic acidmolecule comprising the elements of (a) and (b).
 3. An isolated nucleicacid molecule encoding a carotenoid ketolase and a carotenoidhydroxylase, said isolated nucleic acid molecule comprising: a) anucleic acid fragment encoding a carotenoid ketolase having the aminoacid sequence as represented by SEQ ID NO: 17 and a nucleic acidfragment encoding a carotenoid hydroxylase enzyme having the amino acidsequence as represented by SEQ ID NO: 19; or (b) an isolated nucleicacid molecule completely complementary to the nucleic acid fragment of(a).
 4. An isolated nucleic acid molecule encoding a carotenoid ketolaseand a carotenoid hydroxylase, said isolated nucleic acid moleculecomprising: (a) a nucleic acid fragment encoding a carotenoid ketolasehaving the amino acid sequence as represented by SEQ ID NO: 21; and anucleic acid fragment encoding a carotenoid hydroxylase enzyme havingthe amino acid sequence as represented by SEQ ID NO: 23; or (b) anisolated nucleic acid molecule completely complementary to the nucleicacid fragment of (a).
 5. (canceled)
 6. A transformed host cellcomprising the isolated nucleic acid molecule of any of claims 1 or 3.7. The transformed host cell of claim 6 wherein the host cell isselected from the group consisting of bacteria, yeast, filamentousfungi, algae, and green plants.
 8. The transformed host cell of claim 7wherein the host cell is selected from the group consisting ofAspergillus, Trichoderma, Saccharomyces, Pichia, Phaffia, Candida,Hansenula, Salmonella, Bacillus, Acinectorbacter, Zymomonas,Agrobacterium, Erythrobacter, Chloroborium, Chromatium, Flavobacterium,Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium,Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia,Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylomicrobium, Methylocystis,Alcaligenes, Synechocystis, Methanomonas, Synechococcus, Anabeana,Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus. 9.(canceled)
 10. The transformed host cell of claim 8 wherein the hostcell is Methylomonas sp. 16a having the designation ATCC PTA
 2402. 11.The transformed host cell of claim 7 where the host cell is selectedfrom the group consisting of soybean, rapeseed (Brassica napus, B.campestris), pepper, sunflower (Helianthus annus), cotton (Gossypiumhirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago sativa),wheat (Triticum sp), barley (Hordeum vulgare), oats (Avena sativa, L),sorghum (Sorghum bicolor), rice (Oryza sativa), Arabidopsis, cruciferousvegetables (broccoli, cauliflower, cabbage, parsnips, etc.), melons,carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts,grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye,flax, hardwood trees, softwood trees, and forage grasses.
 12. A methodfor the production of astaxanthin comprising: a) providing a transformedhost cell that produces β-carotene and which comprises the chimeric genecluster of claim 5 encoding at least one carotenoid ketolase enzyme aidat least one carotenoid hydroxylase enzyme; and b) growing thetransformed host cell of (a) under suitable conditions wherebyastaxanthin is produced.
 13. A method according to claim 12, whereinafter step (b) the astaxanthin is optionally isolated.
 14. A methodaccording to claim 12 wherein the carotenoid ketolase is a CrtO ketolaseand the carotenoid hydroxylase is a CrtZ hydroxylase.
 15. A methodaccording to claim 12 wherein the host cell is selected from the groupconsisting of bacteria, yeast, filamentous fungi, algae, and greenplants.
 16. A method according to claim 15 wherein the transformed hostcell is selected from the group consisting of Aspergillis, Trichoderma,Saccharomyces, Pichia, Phaffia, Candida, Hansenula, Salmonella,Bacillus, Acinectorbacter, Zymomonas, Agrobacterium, Erythrobacter,Chloroborium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter,Rhodococcus, Streptomyces, Brevibacteriurn, Corynebacteria,Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoca, Pseudomonas,Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus,Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis,Methanomonas, Synechococcus, Anabeana, Thiobacillus, Methanobacterium,Klebsiella, and Myxococcus.
 17. A method according to claim 16 whereinthe transformed host cell is Methylomonas sp. 16a having the designationATCC PTA
 2402. 18. A method according to claim 15 wherein the host cellis selected from the group consisting of soybean, rapeseed (Brassicanapus, B. campestris), pepper, sunflower (Helianthus annus), cotton(Gossypium hirsutum), corn, tobacco (Nicotiana tabacum), alfalfa(Medicago saliva), wheat (Triticum sp), barley (Hordeum vulgare), oats(Avena sativa, L), sorghum (Sorghum bicolor), rice (Oryza saliva),Arabidopsis, cruciferous vegetables (broccoli, cauliflower, cabbage,parsnips, etc.), melons, carrots, celery, parsley, tomatoes, potatoes,strawberries, peanuts, grapes, grass seed crops, sugar beets, sugarcane, beans, peas, rye, flax, hardwood trees, softwood trees, and foragegrasses.
 19. A method of altering astaxanthin biosynthesis in anorganism comprising: a) providing a host cell capable of producingastaxanthin; b) introducing into said host cell the nucleic acidmolecule of claim 5; and c) growing the host cell of (b) underconditions whereby the nucleic acid molecule is expressed andastaxanthin biosynthesis is altered.
 20. A method according to claim 19wherein the expression of the nucleic acid molecule of claim 5 isupregulated.
 21. A method according to claim 19 wherein the host cell ofstep (c) comprises astaxanthin at a concentration of about 3% to about20% of the total carotenoids produced.
 22. A method according to claim20 wherein the nucleic acid molecule of claim 5 is expressed on amulticopy plasmid
 23. A method according to claim 20 wherein the nucleicacid molecule of claim 5 is operably linked to an inducible or regulatedpromoter.
 24. A method according to claim 19 wherein the nucleic acidmolecule of claim 5 is are down-regulated.
 25. A method to generate andidentify nucleic acid molecules encoding a carotenoid ketolase and acarotenoid hydroxylase having improved astaxanthin biosynthesis activitycomprising: a) providing a host cell capable of producing β-carotene; b)providing a starting pair of genes comprising a carotenoid ketolase geneand a carotenoid hydroxylase; c) exposing said starting pair of genessimultaneously to mutational conditions in vitro to form a mutated genepair; wherein at least one nucleotide has been altered in either one orboth of said carotenoid ketolase gene or said carotenoid hydroxylasegene; d) operably linking the mutated gene pair to a suitable regulatorysequence; e) transforming the host cell of step a) with the mutated genepair from step d) to produce a recombinant host cell; f) growing therecombinant host cell under conditions whereby astaxanthin is produced;g) measuring the amount of astaxanthin produced in step f) and selectingthose transformants having increased astaxanthin production relative tothe level of astaxanthin produced by the starting pair of genes in thehost cell; and h) identifying the mutated gene pair from the selectedtransformants which have increased astaxanthin biosynthesis activity.26. A method according to claim 25 wherein the mutate gene pair isoptionally isolated after step (h).
 27. A method according to claim 25wherein steps (e)-(h) are repeated.
 28. A method according to claim 27wherein the mutational condition is error-prone PCR.